Methods and compositions involving (s)-bucindolol

ABSTRACT

Disclosed is bucindolol substantially free of its R-stereoisomer. Also disclosed are pharmaceutical compositions that include bucindolol substantially free of its R-stereoisomer or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. Also disclosed are methods of treating a patient that involve administering to the patient a therapeutically effective amount of a composition of the present invention.

The present application is a divisional of U.S. patent application Ser.No. 15/268,137, filed Sep. 16, 2016, which is a continuation of U.S.patent application Ser. No. 14/590,661, filed Jan. 6, 2015, now U.S.Pat. No. 9,446,023, which is a divisional of U.S. patent applicationSer. No. 13/056,916, filed May 4, 2011, now U.S. Pat. No. 8,946,284,issued Feb. 3, 2015, which is a national phase application under 35U.S.C. § 371 of International Application No. PCT/US2009/032144, filedJan. 27, 2009, which claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 61/085,586, filed Aug. 1, 2008, each ofwhich are hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates generally to the fields of pharmacologyand clinical medicine. More specifically, the present invention isdirected to pharmaceutical compositions and methods for treatment ofdisease in humans that concern S-bucindolol.

2. Description of Related Art

The human endothelium has an essential role in regulating arterial bloodflow and preserving normal vascular physiology. Important activities ofthe endothelium are mediated by the production of signaling molecules,especially nitric oxide (NO). Endothelial dysfunction, on the otherhand, is linked to atherosclerosis and its clinical manifestations(coronary artery disease, heart failure) (Harrison et al., 1987; Liao,1998; Oemar et al., 1998). Key risk factors for atherosclerosis,including dyslipidemia, smoking and diabetes, can be specifically linkedto abnormalities in NO-mediated endothelial dilation (Harrison et al.,1987; Liao, 1998; Oemar et al., 1998). In addition, a reduction in NObioavailability contributes to elevated vascular resistance and loss ofsensitivity to stimuli of vasodilation, hallmark features ofhypertension (Paniagua et al., 2001; Panza et al., 1990; Taddei et al.,1993).

Beyond vasodilation, NO has well-characterized vascular benefits,including inhibition of smooth muscle cell proliferation and migration,adhesion of leukocytes to the endothelium, and platelet aggregation(Harrison, 1997). In patients at higher risk for cardiovascular diseaseand its clinical consequences (e.g., African Americans), there is alsoevidence for reduced NO-mediated vasodilation associated with increasedsuperoxide generation in endothelial cells (Campia et al., 2002;Kalinowski et al., 2004; Stein et al., 1997). Thus, agents that directlystimulate NO release may have important therapeutic advantages in theprevention and treatment of cardiovascular disease.

Hispanics are the largest and fastest-growing minority group in theUnited States, and Mexican Americans are the largest sub-group ofHispanics. Epidemiologic studies indicate that Mexicans have higherrates of coronary heart disease (CHD) risk equivalents, including type 2diabetes mellitus, metabolic syndrome and some primary forms ofdyslipidemia (Stern et al., 1991; Aguilar-Salinas et al., 2001;Aguilar-Salinas et al., 2003). By the age of 50, epidemiologic studiesindicate that 28% of men and 21% of women in Mexico already exhibit someform of dyslipidemia (Aguilar-Salinas et al.,2001). In the San AntonioHeart Study, it was reported that after adjusting for age and gender,U.S.-born Mexican Americans were 1.4 times as likely to die ofcardiovascular (CV) disease as non-Hispanic whites (Hunt et al., 2002).For cardiovascular disease, U.S.-born Mexican Americans were 1.7 timesmore likely to die from CVD and 1.9 times more likely to die from CHDthan non-Hispanic whites (Hunt et al., 2002). To understand the basisfor this enhanced risk, a recent study has identified genetic variantsthat confer higher susceptibility to dyslipidemia, but more studies areneeded in this area (Aguilar-Salinas et al., 2003; Huerta-Vazquez etal., 2005). There are also well known environmental factors thatcontribute to higher risk in individual Mexican Americans, including ahigh-fat and high-calorie diet, tobacco use, alcohol consumption andsedentary lifestyle.

Hypertension is a risk factor that is less likely treated and controlledamong Hispanics, as compared to the overall U.S. population. This was akey finding from the National Health and Nutrition Examination Surveys(NHANES) for 1999-2002. This report has identified racial/ethnicdisparities in the awareness of, treatment for, and control ofhypertension. NHANES is a stratified, multistage probability sample ofthe civilian, non-institutionalized U.S. population. During 1999-2002,the age-adjusted prevalence of hypertension in the study population was28.6% (CI=26.8%-30.4%). The prevalence of hypertension increased withage, as expected, and was higher among women than men. Among adults withhypertension, the proportion aware of having this condition was 70.3%among non-Hispanic blacks, 62.9% among non-Hispanic whites, but only49.8% among Mexican Americans. The age-adjusted proportion that reportedtreatment was 55.4% among non-Hispanic blacks, 48.6% among non-Hispanicwhites, and only 34.9% among Mexican Americans. Only 29% of U.S. adultswith hypertension had controlled BP levels (<140/90 mmHg). Theproportion with controlled BP was similar among non-Hispanic blacks(29.8%) and non-Hispanic whites (29.8%) but substantially lower amongMexican Americans (17.3%). These findings indicate the challenge ofeffectively treating and controlling hypertension in the rapidly growingHispanic and Mexican American population.

While epidemiologic studies indicate a higher risk of CV disease andlower BP control among Mexican Americans, the underlying pathophysiologyis not well understood. Studies in other high-risk populations, such asAfrican Americans, indicate that the higher risk is related to decreasedresponsiveness of conductance vessels to both endogenous and exogenousstimulants of NO, as compared with age-matched whites (Campia et al.,2002). To understand the basis for this difference, it was reported thatthere is lower bioavailability of NO from endothelium of blackAmericans, despite much higher levels of endothelial-dependent NOsynthase (eNOS) (Kalinowski et al., 2004). The cellular basis for thisparadox was the finding that excessive O₂ ⁻ generation byNAD(P)H-oxidase and uncoupled eNOS resulted in the loss of functional NOdue to its reactivity with O₂ ⁻, resulting in peroxynitrite (ONOO⁻)formation, a potent oxidant with the capacity to produce adversebiological effects (Kalinowski et al., 2004).

Thus, there is the need for improved therapy of hypertension and CVdisease, particularly among racial groups where there is a highprevalence of these diseases.

SUMMARY OF THE INVENTION

The present invention relates to the finding that the S-stereoisomer ofbucindolol (S-bucindolol) has a greater capacity of inducing cells togenerate NO compared to bucindolol racemate while simultaneouslyreducing ONOO⁻ production. The present invention is also related to thefinding that S-bucindolol demonstrates particularly favorable activityin racial groups including African Americans, Mexican Americans, andnon-Hispanic whites.

Certain embodiments of the present invention concern methods andcompositions involving bucindolol(2-(3-(1-(1H-indol-3-yl)-2-methylpropan-2-ylamino)-2-hydroxypropoxy)benzonitrile),substantially free of its R-stereoisomer. A composition is“substantially free” of R-bucindolol if it includes a mixture ofS-bucindolol and (optionally) R-bucindolol wherein the weight ofR-bucinolol, if present, is no more than about 20% of the total weightof S-bucindolol and R-bucindolol in the composition. In someembodiments, the composition may contain no more than about 20, 19, 18,17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4.9, 4.8, 4.7, 4.6, 4.5,4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1,3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7,1.6, 1.5, 1.4, 1.3, 1.2, 1.1, or 1.0% or any range derivable therein byweight of R-bucindolol relative to the total weight of S-bucindolol andR-bucindolol in the composition. In some particular embodiments, thecomposition that is substantially free of R-bucindolol contains no morethan about 20% by weight of R-bucindolol relative to the total weight ofS-bucindolol and R-bucindolol in the composition. In more particularembodiments, the composition contains no more than about 10% by weightof R-bucindolol relative to the total weight of S-bucindolol andR-bucindolol in the composition. In more particular embodiments, theinventive composition contains no more than about 10% of R-bucindololrelative to the total weight of S-bucindolol and R-bucindolol in thecomposition. In even more particular embodiments, the inventivecomposition contains no more than about 1% of R-bucindolol relative tothe total weight of S-bucindolol and R-bucindolol in the composition.

In some embodiments of the invention, it is contemplated that bucindololincludes pharmaceutically acceptable salts of bucindolol. Thus, forexample, a composition comprising S-bucindolol may include apharmaceutically acceptable salt of S-bucindolol. In particularembodiments of the present invention, embodiments include bucindolol orpharmaceutical compositions of bucindolol that do not include anypharmaceutically acceptable salts of bucindolol.

In certain embodiments of the present invention, the bucindolol in thecomposition is substantially purified. “Substantially purified” as setforth herein refers to a composition comprising bucindolol wherein thecomposition includes at least about 80% S-bucindolol. In someembodiments, the composition includes at least about 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.2, 99.2,99.3, 99.4, 99.5, 99.6, 99.7, 99.8, or 99.9% by weight of S-bucindololrelative to total bucindolol in the composition.

The pharmaceutical composition may optionally include one or moreadditional pharmaceutical agents. Any pharmaceutical agent iscontemplated for inclusion in the compositions. Examples of specificagents are set forth in the specification below. In some embodiments,the pharmaceutical agent is an agent that can be applied in thetreatment or prevention of a cardiovascular disease, a neurologicaldisease, an infectious disease, an inflammatory disease, a neoplasm, agastrointestinal disease, a genitourinary disease, a pulmonary disease,or an immune disease. In some embodiments the pharmaceutical agent is anadditional β-adrenergic receptor blocker. Non-limiting examples ofbeta-adrenergic receptor blockers include AC 623, acebutolol,alprenolol, amosulalol, arotinolol, atenolol, befunolol, betaxolol,bevantolol, bisoprolol, bopindolol, bucumolol, bufetolol, bufuralol,bunitrolol, bupranolol, butidrine hydrocholoride, butofilolol,carazolol, carteolol, carvedilol, celiprolol, cetamolol, cloranolol,dilevalol, esmolol, indenolol, labetalol, landiolol, levobunolol,mepindolol, metipranolol, metoprolol, moprolol, nadolol, nadoxolol,nebivolol, nifenalol, nipradilol, oxprenolol, penbutolol, pindolol,practolol, pronethalol, propranolol, sotalol, sulfinalol, talinolol,tertatolol, tilisolol, timolol, toliprolol, and xibenolol.

In other embodiments, the composition may include a nitric oxide (NO)enhancing agent. Examples of NO enhancing agents are well known to thoseof ordinary skill in the art. Examples of such agents include a RASinhibitor, a statin, a PDE5 inhibitor, a NO-conjugated drug, or adiazeniumdiolate. Non-limiting examples of RAS inhibitors includecaptopril, cilazapril, enalapril, fosinopril, lisinopril, quinapril,ramapril, zofenopril, candesartan cilexetil, eprosartan, irbesartan,losartan, tasosartan, tehnisartan, and valsartan, or a pharmaceuticallyacceptable salt thereof. Non-limiting examples of statins includeatorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatincalcium, and simvastatin. Non-limiting examples of NO-conjugated drugsinclude S—NO-glutathione, NO-naproxen, NO-aspirin, NO-ibuprofen,NO-Diclofenac, NO-Flurbiprofen, NO-Ketoprofen, NO-releasing compound-7,NO-releasing compound-5, NO-releasing compound-12, or NO-releasingcompound-18. Other examples of NO enhancing agents include L-arginine,arginine alpha-ketoglutarate, GEA 3175, sodium nitroprusside, glyceryltrinitrate, S-nitroso-N-acetyl-penicillamine, nitroglycerin, anddiethylamine NONOate. Information concerning NO generating compounds fortreating hypertension and atherosclerosis can be found in U.S. Pat. Nos.7,396,829, 7,348,319, 7,155,284, 7,052,695, 6,358,536, and 5,208,233,each of which is herein specifically incorporated by reference.Information regarding nebivolol as an NO-enhancing agent can be found inU.S. Pat. No. 7,138,430, herein specifically incorporated by reference.

The invention also concerns methods of treating a patient involvingadministering to a patient a therapeutically effective amount of acomposition comprising bucindolol wherein the composition issubstantially free of the R-stereoisomer of bucindolol. The compositioncan be any of those compositions set forth above.

In certain embodiments, the patient self-identifies as a Caucasian. Inmore particular embodiments, the patient self-identifies as anon-Hispanic white. In other embodiments, the patient self-identifies asan individual of African descent. In still further embodiments, thepatient self-identifies as a Hispanic. In more particular embodiments,the patient self-identifies as a Mexican American. In some embodimentsthe patient has a disease or condition such that the patient is in needof a NO enhancing agent. For example, the patient may have a headache,hypoxic respiratory failure, pulmonary hypertension, right ventricularheart failure, congestive heart failure, respiratory distress syndrome,impotence, hypertension, angina, myocardial infarction, or cardiacarrhythmia.

In some embodiments, the patient is in need of a beta-blocker. Forexample, the patient may be a patient in need of treatment or preventionof hypertension, angina, myocardial infarction, mitral valve prolapse,cardiac arrhythmia, congestive heart failure, hypertrophic obstructivecardiomyopathy, acute dissecting aortic aneurysm, portal hypertension,anxiety disorder, glaucoma, migraine headache, migraine prophylaxis,tremor due to anxiety, tremor due to hyperthyroidism, essential tremor,pheochromocytoma, or hyperhidrosis.

The method may optionally involve administering to the patient asecondary form of therapy. The secondary form of therapy may be any typeof therapy. For example, the secondary form of therapy may be apharmaceutical agent or a surgical procedure. Non-limiting examples ofsurgical procedures include angioplasty, valve replacement surgery,heart transplant, coronary artery bypass grafting, and peripheralvascular surgery. The secondary therapy may be administered prior to,concurrently with, or following administration of the therapeuticcompositions set forth herein. In particular embodiments, the secondarytherapy is a pharmaceutical agent. The pharmaceutical agent may beadministered separately from the composition of the present invention,or may be included as a component of a composition as set forth herein.Examples of such pharmaceutical agents are set forth above and elsewherein this specification.

Administration of the pharmaceutical compositions set forth herein maybe by any method known to those of ordinary skill in the art. Examplesinclude, but are not limited to, oral, intravenous, intramuscular,intra-arterial, intramedullary, intrathecal, intraventricular,intradermal, intratracheal, intravesicular, intraocular, transdermal,subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual,or rectal administration. Further details on techniques for formulationand administration may be found in the specification below.

In some embodiments, the method further includes contacting the patientwith a medical device that includes S-bucindolol. For example, themedical device may include a coating that includes S-bucindolol, amatrix that includes S-bucindolol, or a reservoir that includes atherapeutic composition as set forth above. The device may be insertedinto the patient temporarily or implanted in the patient or placed on abody surface of the patient. Examples of such body surfaces include skinsurfaces or mucosal surfaces.

The medical device may be any medical device known to those of ordinaryskill in the art. Non-limiting examples of such medical devices includea stent, a graft, a heart valve, a filter, a catheter, a coil, a meshrepair material, a plate, a rod, a screw, or a suture.

The present invention also concerns methods of increasing the capacityof a cell to generate NO, involving contacting the cell with acomposition that includes bucindolol substantially free of itsR-stereoisomer or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier. The cell may be any type of cell.In particular embodiments the cell is an endothelial cell, an epithelialcell, or a stem cell. In certain embodiments, the cell is a cell that isin a patient or an animal. Non-limiting examples of animals include amouse, a rat, a rabbit, a cat, a dog, a horse, a sheep, a goat, a cow,or a primate.

The invention also concerns medical devices that include a coating, amatrix, or a chamber, wherein the coating, matrix, or chamber includesbucindolol substantially free of the R-stereoisomer. Non-limitingexamples of such medical devices include a stent, a graft, a heartvalve, a filter, a catheter, a coil, a mesh repair material, a plate, arod, a screw, and a suture. An example of a type of filter is aninferior vena caval filter. An example of a type of catheter is a druginfusion catheter. An example of a type of coil is an embolic coil.

It is specifically contemplated that any limitation discussed withrespect to one embodiment of the invention may apply to any otherembodiment of the invention. Furthermore, any composition of theinvention may be used in any method of the invention, and any method ofthe invention may be used to produce or to utilize any composition ofthe invention.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativeare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device and/ormethod being employed to determine the value.

As used herein the specification, “a” or “an” may mean one or more,unless clearly indicated otherwise. As used herein in the claim(s), whenused in conjunction with the word “comprising,” the words “a” or “an”may mean one or more than one. As used herein “another” may mean atleast a second or more.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

The following figures form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Schematic diagram of a NO nanosensor placed in close proximityto the surface of a single endothelial cell. The nanosensor measures thelevels of NO, O₂ ⁻, and ONOO⁻ from the intact endothelium in real time.The sensors are made by depositing a sensing material on the tip ofcarbon fiber with a diameter of about 0.5 μm. The fibers are sealed withnonconductive epoxy and electrically connected to wires (gold, copper)with conductive silver epoxy.

FIG. 2. CaI-stimulated NO release in HUVECs isolated from non-Hispanicwhite, Mexican American and African American donors. Values are reportedas mean±S.D. (N=5). *p<0.01 versus Non-Hispanic white controls (ANOVADunnett multiple comparisons test; Overall ANOVA: p<0.0001; F=47.375).

FIG. 3. Effects of bucindolol, (S)-bucindolol, (R)-bucindolol andatenolol on CaI-stimulated NO release in HUVECs isolated fromnon-Hispanic white donors. Values are reported as mean±S.D. (N=4-5).*p<0.001 and ^(†)p<0.05 versus control; ^(B)p<0.01 versus cognatebucindolol treatment; ^(S)p<0.001 versus cognate (S)-bucindololtreatment; and ^(R)p<0.001 versus cognate (R)-bucindolol treatment(ANOVA Student-Newman-Keuls multiple comparisons test; Overall ANOVA:p<0.0001; F=31.062). Abbreviations: (S)-Bucin=(S)-Bucindolol;(R)-Bucin=(R)-Bucindolol.

FIG. 4 Effects of bucindolol, (S)-bucindolol, (R)-bucindolol andatenolol on CaI-stimulated NO release in HUVECs isolated fromMexican-American donors. Values are reported as mean±S.D. (N=4-5).*p<0.001 versus control; ^(B)p<0.001 versus cognate bucindololtreatment; ^(S)p<0.001 versus cognate (S)-bucindolol treatment; and^(R)p<0.001 versus cognate (R)-bucindolol treatment (ANOVAStudent-Newman-Keuls multiple comparisons test; Overall ANOVA: p<0.0001;F=19.458). Abbreviations: (S)-Bucin=(S)-Bucindolol;(R)-Bucin=(R)-Bucindolol.

FIG. 5 Effects of bucindolol, (S)-bucindolol, (R)-bucindolol andatenolol on CaI-stimulated NO release in HUVECs isolated from AfricanAmerican donors. Values are reported as mean±S.D. (N=4-5). *p<0.001 and^(†)p<0.05 versus control; ^(B)p<0.05 versus cognate bucindololtreatment; ^(S)p<0.001 versus cognate (S)-bucindolol treatment; and^(R)p<0.01 versus cognate (R)-bucindolol treatment (ANOVAStudent-Newman-Keuls multiple comparisons test; Overall ANOVA: p<0.0001;F=21.419. Abbreviations: (S)-Bucin=(S)-Bucindolol;(R)-Bucin=(R)-Bucindolol.

FIG. 6 CaI-stimulated ONOO⁻ release in HUVECs isolated from non-Hispanicwhite, Mexican American and African American donors. Values are reportedas mean±S.D. (N=5). *p<0.01 versus control (ANOVA Dunnett multiplecomparisons test; Overall ANOVA: p<0.0001; F=55.340).

FIG. 7 Effects of bucindolol, (S)-bucindolol, (R)-bucindolol andatenolol on CaI-stimulated ONOO⁻ release in HUVECs isolated fromnon-Hispanic white donors. Values are reported as mean±S.D. (N=4-5).*p<0.05, ^(†)p<0.01 and _(§)p<0.001 versus control; ^(B)p<0.05 versuscognate bucindolol treatment; ^(S)p<0.01 versus cognate (S)-bucindololtreatment; and ^(R)p<0.05 versus cognate (R)-bucindolol treatment (ANOVAStudent-Newman-Keuls multiple comparisons test; Overall ANOVA: p<0.0001;F=7.575). Abbreviations: (S)-Bucin=(S)-Bucindolol;(R)-Bucin=(R)-Bucindolol.

FIG. 8 Effects of bucindolol, (S)-bucindolol, (R)-bucindolol andatenolol on CaI-stimulated ONOO⁻ release in HUVECs isolated from MexicanAmerican donors. Values are reported as mean±S.D. (N=4-5). *p<0.05,^(†)p<0.01 and ^(§)p<0.001 versus control; ^(B)p<0.05 versus cognatebucindolol treatment; ^(S)p<0.05 versus cognate (S)-bucindololtreatment; and ^(R)p<0.05 versus cognate (R)-bucindolol treatment (ANOVAStudent-Newman-Keuls multiple comparisons test; Overall ANOVA: p<0.0001;F=15.481). Abbreviations: (S)-Bucin=(S)-Bucindolol;(R)-Bucin=(R)-Bucindolol.

FIG. 9 Effects of bucindolol, (S)-bucindolol, (R)-bucindolol andatenolol on CaI-stimulated ONOO⁻ release in HUVECs isolated from AfricanAmerican donors. Values are reported as mean±S.D. (N=4-5). *p<0.05,^(†)p<0.01 and ^(§)p<0.001 versus control; ^(B)p<0.001 versus cognatebucindolol treatment; ^(S)p<0.05 versus cognate (S)-bucindololtreatment; and ^(R)p<0.01 versus cognate (R)-bucindolol treatment (ANOVAStudent-Newman-Keuls multiple comparisons test; Overall ANOVA: p<0.0001;F=14.628). Abbreviations: (S)-Bucin=(S)-Bucindolol;(R)-Bucin=(R)-Bucindolol.

FIG. 10 CaI-stimulated NO/ONOO⁻ release ratio in HUVECs isolated fromnon-Hispanic white, Mexican American, and African American donors.Values are reported as mean±S.D. (N=5). *p<0.01 versus control (ANOVADunnett multiple comparisons test; Overall ANOVA: p<0.0001; F=79.897).

FIG. 11 Effects of bucindolol, (S)-bucindolol, (R)-bucindolol andatenolol on CaI-stimulated NO/ONOO⁻ release in HUVECs isolated fromnon-Hispanic white donors. Values are reported as mean±S.D. (N=4-5).*p<0.001, and ^(†)p<0.05 versus control; ^(B)p<0.01 versus cognatebucindolol treatment; ^(S)p<0.01 versus cognate (S)-bucindololtreatment; and ^(R)p<0.01 versus cognate (R)-bucindolol treatment (ANOVAStudent-Newman-Keuls multiple comparisons test; Overall ANOVA: p<0.0001;F=21.782). Abbreviations: (S)-Bucin=(S)-Bucindolol;(R)-Bucin=(R)-Bucindolol.

FIG. 12 Effects of bucindolol, (S)-bucindolol, (R)-bucindolol andatenolol on CaI-stimulated NO/ONOO⁻ release in HUVECs isolated fromMexican American donors. Values are reported as mean±S.D. (N=4-5).*p<0.001, and ^(†)p<0.01 versus control; ^(B)p<0.05 versus cognatebucindolol treatment; ^(S)p<0.05 versus cognate (S)-bucindololtreatment; and ^(R)p<0.05 versus cognate (R)-bucindolol treatment (ANOVAStudent-Newman-Keuls multiple comparisons test; Overall ANOVA: p<0.0001;F=29.540). Abbreviations: (S)-Bucin=(S)-Bucindolol;(R)-Bucin=(R)-Bucindolol.

FIG. 13 Effects of bucindolol, (S)-bucindolol, (R)-bucindolol andatenolol on CaI-stimulated NO/ONOO⁻ release in HUVECs isolated fromAfrican American donors. Values are reported as mean±S.D. (N=4-5).*p<0.001, and ^(†)p<0.01 versus control; ^(B)p<0.01 versus cognatebucindolol treatment; ^(S)p<0.05 versus cognate (S)-bucindololtreatment; and ^(R)p<0.001 versus cognate (R)-bucindolol treatment(ANOVA Student-Newman-Keuls multiple comparisons test; Overall ANOVA:p<0.0001; F=30.266). Abbreviations: (S)-Bucin=(S)-Bucindolol;(R)-Bucin=(R)-Bucindolol.

FIG. 14A, 14B. 14A—Competition curve between S- or R-bucindolol in humanLV membranes that are 86% β₁. AR and genotypically 389 Arg/Arg. Therespective K_(i)s are 0.49 nM and 14.0 nM for the S- and R-isomers.14B—Competition curve between S- or R-bucindolol in human LV membranesthat are 83% β₁. AR and genotypically 389 Gly/Gly. The respective K_(i)sare 0.59 nM and 26.3 nM for the S- and R-isomers.

FIG. 15. ¹H NMR spectrum of (R)-bucindolol (sample in DMSO-d₆); 300 MHz.

FIG. 16. ¹H NMR spectrum of (S)-bucindolol (sample in DMSO-d₆); 300 MHz.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Bucindolol is a nonselective β-blocking agent with mild vasodilatoryproperties. The present invention is in part based on the finding thatthe mechanism of vasodilation with bucindolol involvesendothelial-dependent nitric oxide (NO) release, and that it iseffective in higher risk racial groups, such as African Americans andMexican Americans. In this regard, the inventors examined the effects ofbucindolol and its separate enantiomers on endothelial-dependent NO andnitroxidative stress (peroxynitrite) release in cells from healthywhite, African American and Mexican American donors. The effects ofbucindolol and its enantiomers were compared to another β₁-selectiveantagonist, atenolol. It was found that bucindolol had a dual effect onendothelial function by increasing the capacity of cells to generate NOwhile simultaneously reducing ONOO⁻ production in a stereoselectivemanner. The favorable activity of bucindolol on the NO/ONOO⁻ ratio wassignificant and highly dose-dependent in three different racial groups,including African Americans, Mexican Americans and non-Hispanic whites.The activity of (S)-bucindolol was superior to bucindolol racemate butnot reproduced by atenolol under identical conditions.

A. BETA BLOCKERS AND BUCINDOLOL

1. β-Blockers

Treatment for heart failure has involved targeting adrenergic receptors(AR).

There are at least nine sub-types of adrenergic receptors (Dohlman etal., 1991; and Liggett et al., 1993), of which at least three sub-typesare β-adrenergic receptors.

The β₁ adrenergic receptor (β₁AR) is the principle subtype expressed oncardiac myocytes. The human heart expresses both the β₁AR and the β₂ARsubtypes (Bristow et al, 1986; Bristow et al., 1988). Each receptormediates positive inotropic and chronotropic responses to endogenouscatecholamines and exogenously administered agonists (Bristow et al.,1986; Brodde et al., 1986; Brodde et al., 1992).

The β₁AR triggers the heart's contractile response when activated, as itis by norepinephrine. In addition, the β₁ receptor has a central role inthe progression of cardiomyopathy and other disease pathways. Increasedactivation of this receptor and its associated myopathic and arrhythmicpathways plays a major role in the natural history of heart failure.Once the cardiomyopathic process has begun, chronic activation ofβ₁-adrenergic receptors can accelerate disease progression, as thefailing heart tries to compensate for its impaired functioning byreleasing more norepinephrine and increasing β₁-receptor signaling. Thetheory of β-receptor blockade rests in part on counteracting thiscardiomyopathic pathway by blocking the β₁-receptor and reducingnorepinephrine signaling.

The β₁ adrenergic receptor has been cloned and sequenced (Frielle etal., 1987). The gene has been localized to chromosome q24-q26 ofchromosome 10 (Yang-Feng et al., 1990). The human β₁AR has a deducedamino acid sequence of 477 amino acids.

At coding nucleotide position 1165 of the β₁AR gene, either cytosine orguanine can be found in the human population, which results in eitherArg or Gly being encoded at amino acid position 389 (Mason et al.,1999). This position is within an intracellular domain of the receptorthat is involved with coupling to the stimulatory G-protein, Gs. Infibroblasts transfected to express equal levels of the two receptors,the β₁-Arg389 receptor display substantially greater stimulation ofadenylyl cyclase compared to β₁-Gly389 (Mason et al., 1999). A lesscommon polymorphism of the β₁AR, Gly49, has also been identified butthere are discrepant reports as to its functional implications (Rathz etal., 2002; Levin et al., 2002).

The β₁-AR 389Arg/Arg polymorphism is actually the most prevalent form ofthe β₁ adrenergic receptor and is present in about 50% of the U.S.population (slightly less in African-Americans). The other variant ofthis receptor has a glycine (Gly) at the 389 position and is consideredthe wild type only because it was cloned first. Liu et al. (2003) reportfinding that a greater response (in terms of changes in heart rate) tometoprolol was associated with Arg389 compared to Gly389.

While β₁ agonists such as dobutamine, are used for treating acutedeterioration of patients with failing ventricular function, prolongedexposure of the heart from administered agonists, or the elevatedendogenous catecholamine agonists produced by the body, leads toworsening heart failure. Indeed β₁AR and β₂AR become desensitized inheart failure, which is thought to be a mechanism of self-protectionagainst the high levels of catecholamines that exist in heart failure.The administration of β antagonists can improve ventricular function andclinical outcomes, presumably by blocking these deleterious effects ofcatecholamines. And indeed, cardiac βAR expression and function improveduring β blockade treatment of heart failure. The vast majority of thedeleterious effects of catecholamines, and the success of β blockertherapy is due to variants of the β₁AR subtype. (Zhu et al., 2001; andBristow et al., 2003).

β-adrenergic receptor antagonists (also termed β-blockers) have emergedas a major treatment modality in chronic heart failure. Initially theseagents were thought to be contraindicated in heart failure, sinceincreased adrenergic drive was thought to be critical for supporting thefailing heart. In fact, in early experience with the 1^(st) generationcompound propranolol, administration of standard doses was frequentlyassociated with worsening of heart failure (Stephen, 1968). However,using low starting doses and slow up-titration, 2^(nd) generation(selective β₁-blockers) or 3^(rd) generation (nonselectiveβ-blocker-vasodilators) generation compounds have been shown to reversecontractile dysfunction as well as structural and molecular remodeling,and to improve heart failure morbidity and mortality (Bristow, 2000);CIBIS-II Investigators and Committees. The cardiac insufficiencybisoprolol study II: a (CIBIS-II, 1999); MERIT-HF Study Group. Effect ofmetoprolol CR/XL in chronic heart failure: Metoprolol CR/XL RandomizedIntervention Trial in Congestive Heart Failure (MERIT-HF, 1999). Packeret al. (2001); BEST Trial Investigators, (2001); Lowes et al., 2002). Inpart, these beneficial effects are thought to be due to a protection ofthe failing heart, which has limited metabolic and physiologic reserves,from persistent adverse biological effects mediated by elevatednorepinephrine levels found in the syndrome (Bristow, 2000; Cohn et al.,1984; and Liggett, 2001). In addition, β-blockers have been shown topartially reverse the molecular phenotype of heart failure (Lowes etal., 2002), so these agents are capable of both preventing and reversingprogressive myocardial failure and remodeling Eichhorn and Bristow,Circulation 1996).

Bucindolol and metoprolol have some notable differences in theirpharmacologic properties (Bristow, 2000; and Bristow et al., 1997). Inparticular, bucindolol lowers norepinephrine, dilates the peripheralvasculature, and more potently blocks the human β₁-adrenergic receptor.

While a common pharmacologic property of all β-blocking agents that havebeen used to treat HF is that they block the β₁AR, which in the failinghuman heart has been estimated to transduce up to approximately 90% ofthe pathologic adrenergic stimulation (Zhu et al., 2001; and Bristow etal., 2003), the available β-blockers have a number of distinguishingproperties including βAR-subtype selectivity, affinity for α₁AR, partialagonist activity, sympatholysis (Bristow et al., 2004) and vasodilation(Bristow, 2000; and Bristow et al., 1997).

β-blockers have significant structural differences. Moreover, they havedifferent pharmacological properties. Carvedilol, for instance, is anefficient β₁-AR and β₂-AR blocker, as well as an α₁-AR blocker. Incontrast, bucindolol is a weak α₁-AR blocker, and metoprolol andbisoprolol do not block α₁-AR at all. Significantly, bucindolol isunique among β-blockers in its sympatholytic properties, in contrast tocarvedilol, metoprolol, and bisoprolol, which have no such properties.Compared to other β-blocking agents bucindolol uniquely lowers systemicnorepinephrine levels (Lowes et al., 2000; Bristow et al., 1997; BESTNEJM, Bristow, 2004), and is a full agonist for the β₃-adrenergicreceptor (Strosberg, 1997).

2. Bucindolol

Bucindolol is a 3rd generation, β-blocker-vasodilator with the chemicalname and structure of(2-{2-hydroxy-3{{2-(3-indolyl)-1,1-dimethylethyl}amino}propoxy}-benzonitrilehydrochloride). It was first developed for treatment of hypertension,and subsequently for the treatment of heart failure. Because of its lowinverse-agonist and vasodilator properties the nonselective β-blockadeof bucindolol is relatively well tolerated by heart failure patients,and in part for this reason in 1994 bucindolol was selected by the NIHand VA Cooperative Clinical Trials Group to test the hypothesis that aβ-blocker could reduce mortality in advanced heart failure. The test ofthis hypothesis was the BEST Trial, which was conducted between May 31,1995 and Jul. 29, 1999.

The Beta-blocker Evaluation of Survival Trial (“BEST”) was stoppedprematurely on recommendation of the Data and Safety MonitoringCommittee, at a time when the hazard ratio for the primary endpoint ofall-cause mortality was apparently 0.90 (C.I.s 0.78-1.02) (BESTInvestigators, 2001; Domanski et al., 2003). However, the results forthe entire BEST cohort were positive for the high order secondaryendpoint of mortality or heart failure hospitalization, which wasreduced by bucindolol by 19% with a p-value of <0.0001 (Domanski et al.,2003). This endpoint is in fact increasingly viewed as the preferredprimary endpoint for HF pivotal trials.

The reasons why BEST was stopped were 1) confirmation by BEST Trial datagenerated in Class III, non-Black patients of the then recentlypublished information from CIBIS-II (CIBIS Investigators, 1999) andMERIT-HF (MERIT-HF Study Group, 1999) trials that these types of heartfailure patients have a substantial survival benefit from β-blockade, 2)increasing loss of equipoise among investigators, who believed that theefficacy of β-blockade in heart failure had been demonstrated, and 3)inefficacy and trends toward adverse events in subgroups (Class IV andBlacks) that had not been previously investigated in β-blocker heartfailure trials. Further development of bucindolol was then abandonedbecause it was not clear bucindolol could be successfully marketed, evenif approved.

Therefore, in this large survival trial in which the end pointevaluation was overall survival, the BEST clinical trial was terminatedearly because of confirmation of benefit that had recently been shown inother trials, and the inability to extend the efficacy of bucindolol topatient subgroups that had not been previously evaluated in large scaleclinical trials (BEST Investigators, 2001). At that time, there was nosignificant difference in mortality observed between those treated withbucindolol or with a placebo. In distinct contrast to the results ofBEST, similar studies with the β-adrenergic antagonists bisoprolol(termed “CIBIS-II” trial), metoprolol (termed “MERIT-HF” trial), andcarvedilol (termed “COPERNICUS” trial) reported very favorabledifferences (34-35% reductions in mortality) between those treated withthe antagonists and those treated with a placebo. The BEST investigatorsspeculated that one possible explanation for the difference in theresults “may derive from the unique pharmacological properties ofbucindolol.”

In the CIBIS-II trial, the study was also stopped early, but because themortality rates were significantly less in those treated withbisoprolol. CIBIS-II Investigators, 1999. Similarly, in the MERIT-HFstudy with metoprolol, the study was ended prematurely because thepredefined criterion had been met and exceeded. MERIT-HF Study Group,1999. The COPERNICUS study involving carvedilol was also halted earlybecause of the significant benefits observed with treatment. Packer etal., 2001. The BEST investigators noted that their results raisedquestions about the equivalency of β-blockers.

Therefore, there are therapeutic differences between bucindolol andother β-blockers, and there was a significant question regarding thetherapeutic efficacy of bucindolol overall. Consequently, anyrelationship between bucindolol and particular genetic variants was notevident.

The benefit of retrospective analysis based on the genetic datadisclosed herein highlights the unique pharmacologic features ofbucindolol that contribute to its effectiveness in treating heartfailure patients. Two of these features are also instrumental in theinteraction of the drug with the adrenergic receptor gene variants.

The first of these features is sympatholysis, or the ability of a drugto lower adrenergic drive directly (lower norepinephrine levels in bloodand tissue). As noted above, among β-blockers that have been used totreat heart failure, bucindolol is unique in this regard (BEST TrialInvestigators, 2001; Lowes et al., 2000; Bristow et al., 2004). Thesympatholytic effects of bucindolol are likely due to β₂-receptorblockade coupled with not enough α₁-blockade to activate adrenergicdrive. Other properties of bucindolol that could contribute tosympatholysis are nitric oxide generation and β₃-receptor agonism(Strosberg, 1997). These latter two properties, plus or minus weakα₁-receptor blockade, likely account for the mild vasodilator propertiesof bucindolol (Gilbert et al., 1990) which, unlike carvedilol, are notsufficiently powerful to trigger reflex adrenergic activation.

When present in modest amounts, (smaller reductions in norepinephrine)sympatholysis is a favorable property, contributing to the therapeuticanti-adrenergic effect of bucindolol. This is a potentially superiormechanism of action to simple β-blockade, as excess norepinephrine isremoved from the system. Norepinephrine is toxic to heart muscle and inexcess amounts triggers various cardiac disease pathways. However, whenexaggerated, sympatholysis can be harmful, and can increase mortality(Bristow et al. 2004). As discussed below, genetic targeting ofbucindolol allows this property to function only in a favorable manner.

The second pharmacologic property of bucindolol that interacts with apharmacogenetic target is high affinity β₁-receptor blockade(Hershberger et al., 1990; Asano et al., 2001). Bucindolol has highaffinity for human β₁-receptors, as well as for β₂-receptors(Hershberger et al., 1990). In addition, through a non-agonist effect oneither translation or protein turnover, bucindolol lowers β₁-receptordensity (Asano et al., 2001). Because it is so well tolerated,bucindolol can be administered at very high β-blocking doses, and inaddition bucindolol uniquely (compared to carvedilol or metoprolol)inactivates constitutively active β₁ 389Arg/Arg receptors (Liggett etal., 2006; Walsh et al., 2008) and each of these properties contributesto its salutary effects on the high functioning human β₁-receptor389Arg/Arg gene variant (Examples, Mason et al., 1999; Liggett et al.2006). Although bucindolol has intrinsic symapthomimetic activity (ISA)in rat myocardium in functioning human cardiac tissue bucindolol isdevoid of ISA (Bristow et al., 1994; Sederberg et al., 2000; Bristow etal., 1998, Example 7). This can clearly be seen in FIG. 13, panels A andB, where no significant increase in force development occurs in isolatedfailing human right ventricular trabeculae, even in the presence ofsignal transduction augmentation with the diterpene compound forskolin,in either the β₁AR Arg/Arg or Gly carrier genotypes. In contrast, asshown in FIG. 13 panel C, xamoterol as a positive control ISA compoundexhibits an increase in force in both low and high signal transductionactivation in the β₁AR Arg/Arg genotype, but only in the high activationstate rendered by forskolin pretreatment in Gly carriers. Finally, asshown in FIG. 13, in preparations of isolated human heart, bucindololhas unique effects on β₁AR Arg/Arg vs Gly carrier receptors. Underconditions of low levels of signal transduction (low receptoractivation) in the failing heart (Panel A), bucindolol functions as aneutral antagonist (no agonist or inverse agonist activity) at the humanmyocardial β₁Arg/Arg receptor, but when signal transduction is high aswhen adenylyl cyclase is directly activated by forskolin (Panel B),bucindolol functions as an inverse agonist, inactivating the receptor asindicated by a statistically significant slope factor up to the highestconcentration achievable in plasma by therapeutic doses, 10-6 M. No sucheffect occurs in Gly carrier receptors, where bucindolol functions as aninverse agonist in low activation states, and a neutral antagonist inthe presence of forskolin. These data suggest that bucindolol isuniquely effective in antagonizing high activation states of theβ₁389Arg/Arg receptor, the form of the receptor that would be expectedto be the most cardiomyopathic.

These properties are likely reasons for the surprising and unexpectedresults that were observed with the Arg389 genetic variant in the β₁ARand the Del322-325 genetic variant in α_(2c)AR in the context ofbucindolol treatment.

3. Stereoisomers of Bucindolol

Racemic mixtures of bucindolol or its stereoisomers can be obtained fromcommercial sources or can be produced by methods well-known to those ofordinary skill in the art. Commercial sources of bucindolol and itsenantiomers include Knoll AG and Bristol-Myers Squib Co. Informationregarding synthesis of bucindolol can be obtained from any of a varietyof sources known to those of ordinary skill in the art, such as U.S.Pat. No. 6,927,036 and WO 1987003584, each of which is hereinspecifically incorporated by reference.

S-bucindolol can also be prepared by the resolution of racemicmaterials, using conventional means such as fractional crystallization,simple crystallization and chromatography on a chiral substrate,extraction, distillation, column chromatography, high performance liquidchromatography, and the like. Additional information regardingpreparation of S-bucindolol and separation of S-bucindolol from aracemic mixture is discussed in the specification below.

B. PREVENTION AND THERAPY

1. Diseases to be Treated or Prevented

Some embodiments of the present invention concern methods of treating apatient. The patient may have any disease or condition for whichtreatment of S-bucindolol is indicated. For example, the disease orcondition may be one for which treatment with a NO-enhancer isindicated. The disease or condition may be one for which treatment witha beta-blocker is indicated. Examples of such diseases and conditionsare discussed elsewhere in this specification.

“Treatment” and “treating” as used herein refer to administration orapplication of a therapeutic agent to a subject or performance of aprocedure or modality on a subject for the purpose of obtaining atherapeutic benefit of a disease or health-related condition. Forexample, a pharmaceutical composition that includes S-bucindolol may beadministered to a subject to reduce the symptoms of congestive heartfailure.

The term “therapeutic benefit” or “therapeutically effective” as usedthroughout this application refers to anything that promotes or enhancesthe well-being of the subject with respect to the medical treatment ofthis condition. This includes, but is not limited to, a reduction in thefrequency or severity of the signs or symptoms of a disease. Forexample, reducing the symptoms of congestive heart failure may includereducing peripheral edema or increasing exercise tolerance.

Other embodiments of the present invention concern methods of preventinga disease in a patient. “Prevention” and “preventing” are used accordingto their ordinary and plain meaning to mean “acting before” or such anact. In the context of a particular disease or health-related condition,those terms refer to administration or application of an agent, drug, orremedy to a subject or performance of a procedure or modality on asubject for the purpose of blocking the onset of a disease orhealth-related condition. For example, a composition comprisingS-bucindolol may be administered to a patient to prevent onset of amyocardial infarction or to prevent the development of symptomsassociated with congestive heart failure.

2. Routes of Administration

Administration of the pharmaceutical compositions comprisingS-bucindolol set forth herein may be by any number of routes including,but not limited to oral, intravenous, intramuscular, intra-arterial,intramedullary, intrathecal, intraventricular, intradermal,intratracheal, intravesicle, intraocular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectal.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.). In certain embodiments bucindololis formulated for oral administration.

3. Formulations

Where clinical applications are contemplated, pharmaceuticalcompositions will be prepared in a form appropriate for the intendedapplication. Generally, this will entail preparing compositions that areessentially free of pyrogens, as well as other impurities that could beharmful to humans or animals.

One will generally desire to employ appropriate salts and buffers torender delivery vectors stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa patient. Aqueous compositions of the present invention comprise aneffective amount of the vector or cells, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. The phrase“pharmaceutically” or “pharmacologically acceptable” refer to molecularentities and compositions that do not produce adverse, allergic, orother untoward reactions when administered to an animal or a human. Asused herein, “pharmaceutically acceptable carrier” includes solvents,buffers, solutions, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the likeacceptable for use in formulating pharmaceuticals, such aspharmaceuticals suitable for administration to humans. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredients of the present invention, itsuse in therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions, providedthey do not inactivate the vectors or cells of the compositions.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention may be via any common route so longas the target tissue is available via that route. This includes oral,nasal, or buccal. Alternatively, administration may be by intradermal,subcutaneous, intramuscular, intraperitoneal or intravenous injection,or by direct injection into cardiac tissue. Such compositions wouldnormally be administered as pharmaceutically acceptable compositions, asdescribed supra.

The active compounds may also be administered parenterally orintraperitoneally. By way of illustration, solutions of the activecompounds as free-base or pharmacologically acceptable salts can beprepared in water suitably mixed with a surfactant, such ashydroxypropylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations generallycontain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include, forexample, sterile aqueous solutions or dispersions and sterile powdersfor the extemporaneous preparation of sterile injectable solutions ordispersions. Generally, these preparations are sterile and fluid to theextent that easy injectability exists. Preparations should be stableunder the conditions of manufacture and storage and should be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi. Appropriate solvents or dispersion media may contain, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

For oral administration the polypeptides of the present inventiongenerally may be incorporated with excipients and used in the form ofnon-ingestible mouthwashes and dentifrices. A mouthwash may be preparedincorporating the active ingredient in the required amount in anappropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan antiseptic wash containing sodium borate, glycerin and potassiumbicarbonate. The active ingredient may also be dispersed in dentifrices,including: gels, pastes, powders and slurries. The active ingredient maybe added in a therapeutically effective amount to a paste dentifricethat may include water, binders, abrasives, flavoring agents, foamingagents, and humectants.

The compositions of the present invention generally may be formulated ina neutral or salt form. Pharmaceutically-acceptable salts include, forexample, acid addition salts (formed with the free amino groups of theprotein) derived from inorganic acids (e.g., hydrochloric or phosphoricacids, or from organic acids (e.g., acetic, oxalic, tartaric, mandelic,and the like. Salts formed with the free carboxyl groups of the proteincan also be derived from inorganic bases (e.g., sodium, potassium,ammonium, calcium, or ferric hydroxides) or from organic bases (e.g.,isopropylamine, trimethylamine, histidine, procaine and the like.

Upon formulation, solutions are preferably administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations may easily be administeredin a variety of dosage forms such as injectable solutions, drug releasecapsules and the like. For parenteral administration in an aqueoussolution, for example, the solution generally is suitably buffered andthe liquid diluent first rendered isotonic for example with sufficientsaline or glucose. Such aqueous solutions may be used, for example, forintravenous, intramuscular, subcutaneous and intraperitonealadministration. Preferably, sterile aqueous media are employed as isknown to those of skill in the art, particularly in light of the presentdisclosure. By way of illustration, a single dose may be dissolved in 1ml of isotonic NaCl solution and either added to 1000 ml ofhypodermoclysis fluid or injected at the proposed site of infusion, (seefor example, “Remington's Pharmaceutical Sciences” 15th Edition, pages1035-1038 and 1570-1580). Some variation in dosage will necessarilyoccur depending on the condition of the subject being treated. Theperson responsible for administration will, in any event, determine theappropriate dose for the individual subject. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

4. Controlled/Extended/Sustained/Prolonged Release Administration

Another aspect of this invention provides methods of treating patientsby delivering the pharmaceutical compositions set forth herein as acontrolled release formulation. As used herein, the terms “controlled,”“extended,” “sustained,” or “prolonged” release of the composition ofthe present invention will collectively be referred to herein as“controlled release,” and includes continuous or discontinuous, andlinear or non-linear release of the composition of the presentinvention. There are many advantages for a controlled releaseformulation of β-blockers.

a. Tablets

A controlled release tablet suitable for purposes of this invention isdisclosed in U.S. Pat. No. 5,126,145, which is incorporated by referenceherein. This tablet comprises, in admixture, about 5-30% high viscosityhydroxypropyl methyl cellulose, about 2-15% of a water-solublepharmaceutical binder, about 2-20% of a hydrophobic component such as awaxy material, e.g., a fatty acid, and about 30-90% active ingredient.

b. Films

This invention further provides a prophylaxis for or method of treatinga patient following an invasive cardiac procedure comprisingadministering biodegradable, biocompatible polymeric film comprisingS-bucindolol, to a patient. The polymeric films are thin compared totheir length and breadth. The films typically have a uniform selectedthickness between about 60 micrometers and about 5 mm. Films of betweenabout 600 micrometers and 1 mm and between about 1 mm and about 5 mmthick, as well as films between about 60 micrometers and about 1000micrometers, and between about 60 and about 300 micrometers are usefulin the manufacture of therapeutic implants for insertion into apatient's body. The films can be administered to the patient in a mannersimilar to methods used in adhesion surgeries. For example, a β-blocker,such as bucindolol, film formulation can be sprayed or dropped onto acardiac tissue site or artery during surgery, or a formed film can beplaced over the selected tissue site. In an alternative embodiment, thefilm can be used as controlled release coating on a medical device suchas a stent, as is discussed in further detail below.

Either biodegradable or nonbiodegradable polymers may be used tofabricate implants in which the β-blocker is uniformly distributedthroughout the polymer matrix. A number of suitable biodegradablepolymers for use in making the biodegradable films of this invention areknown to the art, including polyanhydrides and aliphatic polyesters,preferably polylactic acid (PLA), polyglycolic acid (PGA) and mixturesand copolymers thereof, more preferably 50:50 copolymers of PLA:PGA andmost preferably 75:25 copolymers of PLA:PGA. Single enantiomers of PLAmay also be used, preferably L-PLA, either alone or in combination withPGA. Polycarbonates, polyfumarates and caprolactones may also be used tomake the implants of this invention.

The amount of the S-bucindolol to be incorporated into the polymericfilms of this invention is an amount effective to show a measurableeffect in treating diseases having similar pathophysiological states,such as but not limited to, heart failure, pheochromocytoma, migraines,cardiac arrhythmias, hypertension, aschemia, cardiomyopathy, and variousanxiety disorders. The composition of the present invention can beincorporated into the film by various techniques such as by solutionmethods, suspension methods, or melt pressing.

c. Transdermal Patch Device

Transdermal delivery involves delivery of a therapeutic agent throughthe skin for distribution within the body by circulation of the blood.Transdermal delivery can be compared to continuous, controlledintravenous delivery of a drug using the skin as a port of entry insteadof an intravenous needle. The therapeutic agent passes through the outerlayers of the skin, diffuses into the capillaries or tiny blood vesselsin the skin and then is transported into the main circulatory system.

Transdermal patch devices that provide a controlled, continuousadministration of a therapeutic agent through the skin are also wellknown in the art. Such devices, for example, are disclosed in U.S. Pat.Nos. 4,627,429; 4,784,857; 5,662,925; 5,788,983; and 6,113,940, whichare all incorporated herein by reference. Characteristically, thesedevices contain a drug impermeable backing layer which defines the outersurface of the device and a permeable skin attaching membrane, such asan adhesive layer, sealed to the barrier layer in such a way as tocreate a reservoir between them in which the therapeutic agent isplaced. In one embodiment of the present invention a formulation of theβ-blocker is introduced into the reservoir of a transdermal patch andused by a patient who is homozygous Arg389 at the β₁AR genes.

5. Medical Devices

Another embodiment contemplates the incorporation of S-bucindolol or acomposition comprising S-bucindolol as set forth herein into a medicaldevice that is then positioned to a desired target location within thebody, whereupon the S-bucindolol elutes from the medical device. As usedherein, “medical device” refers to a device that is introducedtemporarily or permanently into a mammal for the prophylaxis or therapyof a medical condition. These devices include any that are introducedsubcutaneously, percutaneously or surgically to rest within an organ,tissue or lumen. Medical devices include, but are not limited to,stents, synthetic grafts, artificial heart valves, artificial hearts andfixtures to connect the prosthetic organ to the vascular circulation,venous valves, abdominal aortic aneurysm (AAA) grafts, inferior venalcaval filters, catheters including permanent drug infusion catheters,embolic coils, embolic materials used in vascular embolization (e.g.,PVA foams), mesh repair materials, a Dracon vascular particle orthopedicmetallic plates, rods and screws and vascular sutures.

In one embodiment, the medical device such as a stent or graft is coatedwith a matrix. The matrix used to coat the stent or graft according tothis invention may be prepared from a variety of materials. A primaryrequirement for the matrix is that it be sufficiently elastic andflexible to remain unruptured on the exposed surfaces of the stent orsynthetic graft.

6. Dosages

The amount of S-bucindolol or composition comprising S-bucindolol thatis administered or prescribed to the patient can be about, at leastabout, or at most about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250,260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390,400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500 mg of totalbucindolol or S-bucindolol, or any range derivable therein.Alternatively, the amount administered or prescribed may be about, atleast about, or at most about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006,0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08,0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7. 3.8, 3.9, 4.0, 4.1,4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 mg/kg of total bucindolol orS-bucindolol, or any range derivable therein, with respect to the weightof the patient.

When provided in a discrete amount, each intake of S-bucindolol orcomposition comprising S-bucindolol can be considered a “dose.” Amedical practitioner may prescribe or administer multiple doses over aparticular time course (treatment regimen) or indefinitely.

The therapeutic composition may be administered 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80,or more times or any range derivable therein. It is further contemplatedthat the drug may be taken for an indefinite period of time or for aslong as the patient exhibits symptoms of the medical condition for whichthe therapeutic agent was prescribed. Also, the drug may be administeredevery 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24 hours, or 1, 2, 3, 4, 5, 6, 7 days, or 1, 2, 3,4, 5 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more, orany range derivable therein. Alternatively, it may be administeredsystemically over any such period of time and be extended beyond morethan a year.

C. OTHER THERAPEUTIC OPTIONS

In certain embodiments of the invention, methods may involveadministering a beta-blocker that is not bucindolol for the treatment ofa disease or disorder in a subject. These agents may be prescribed oradministered instead of or in addition to bucindolol.

As a second therapeutic regimen, the agent may be administered or takenat the same time as S-bucindolol, or either before or afterS-bucindolol. The treatment may improve one or more symptoms of diseasesuch as providing increased exercise capacity, increased cardiacejection volume, decreased left ventricular end diastolic pressure,decreased pulmonary capillary wedge pressure, increased cardiac outputor cardiac index, lowered pulmonary artery pressures, decreased leftventricular end systolic and diastolic dimensions, decreased left andright ventricular wall stress, decreased wall tension and wallthickness, increased quality of life, and decreased disease-relatedmorbidity and mortality.

In another embodiment, it is envisioned to use S-bucindolol incombination with other therapeutic modalities. Thus, in addition to thetherapies described above, one may also provide to the patient more“standard” pharmaceutical cardiac therapies. Examples of other therapiesinclude, without limitation, other beta blockers, anti-hypertensives,cardiotonics, anti-thrombotics, vasodilators, hormone antagonists,iontropes, diuretics, endothelin antagonists, calcium channel blockers,phosphodiesterase inhibitors, ACE inhibitors, angiotensin type 2antagonists and cytokine blockers/inhibitors, and HDAC inhibitors.

Combinations may be achieved by contacting cardiac cells with a singlecomposition or pharmacological formulation that includes both agents, orby contacting the cell with two distinct compositions or formulations,at the same time, wherein one composition includes the expressionconstruct and the other includes the agent. Alternatively, the therapyusing S-bucindolol may precede or follow administration of the otheragent(s) by intervals ranging from minutes to weeks. In embodimentswhere the other agent and expression construct are applied separately tothe cell, one would generally ensure that a significant period of timedid not expire between the time of each delivery, such that the agentand expression construct would still be able to exert an advantageouslycombined effect on the cell. In such instances, it is contemplated thatone would typically contact the cell with both modalities within about12-24 hours of each other and, more preferably, within about 6-12 hoursof each other, with a delay time of only about 12 hours being mostpreferred. In some situations, it may be desirable to extend the timeperiod for treatment significantly, however, where several days (2, 3,4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse betweenthe respective administrations.

It also is conceivable that more than one administration of eitherS-bucindolol, or the other agent will be desired. In this regard,various combinations may be employed. By way of illustration, where theS-bucindolol is “A” and the other agent is “B”, the followingpermutations based on 3 and 4 total administrations are exemplary:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/AA/B/B/B B/A/B/B B/B/A/BOther combinations are likewise contemplated.

1. Pharmacological Therapeutic Agents

Pharmacological therapeutic agents and methods of administration,dosages, etc., are well known to those of skill in the art (see forexample, the “Physicians Desk Reference”, Klaassen's “ThePharmacological Basis of Therapeutics”, “Remington's PharmaceuticalSciences”, and “The Merck Index, Eleventh Edition”, incorporated hereinby reference in relevant parts), and may be combined with the inventionin light of the disclosures herein. Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject, and suchindividual determinations are within the skill of those of ordinaryskill in the art.

Non-limiting examples of a pharmacological therapeutic agent that may beused in the present invention include an antihyperlipoproteinemic agent,an antiarteriosclerotic agent, an antithrombotic/fibrinolytic agent, ablood coagulant, an antiarrhythmic agent, an antihypertensive agent, avasopressor, a treatment agent for congestive heart failure, anantianginal agent, an antibacterial agent or a combination thereof.

In addition, it should be noted that any of the following may be used todevelop new sets of cardiac therapy target genes as β-blockers were usedin the present examples (see below). While it is expected that many ofthese genes may overlap, new gene targets likely can be developed.

In certain embodiments, administration of an agent that lowers theconcentration of one of more blood lipids and/or lipoproteins, knownherein as an “antihyperlipoproteinemic,” may be combined with acardiovascular therapy according to the present invention, particularlyin treatment of athersclerosis and thickenings or blockages of vasculartissues. In certain aspects, an antihyperlipoproteinemic agent maycomprise an aryloxyalkanoic/fibric acid derivative, a resin/bile acidsequesterant, a HMG CoA reductase inhibitor, a nicotinic acidderivative, a thyroid hormone or thyroid hormone analog, a miscellaneousagent or a combination thereof.

Non-limiting examples of aryloxyalkanoic/fibric acid derivatives includebeclobrate, enzafibrate, binifibrate, ciprofibrate, clinofibrate,clofibrate (atromide-S), clofibric acid, etofibrate, fenofibrate,gemfibrozil (lobid), nicofibrate, pirifibrate, ronifibrate, simfibrateand theofibrate.

Non-limiting examples of resins/bile acid sequesterants includecholestyramine (cholybar, questran), colestipol (colestid) andpolidexide.

Non-limiting examples of HMG CoA reductase inhibitors include lovastatin(mevacor), pravastatin (pravochol) or simvastatin (zocor).

Non-limiting examples of nicotinic acid derivatives include nicotinate,acepimox, niceritrol, nicoclonate, nicomol and oxiniacic acid.

Non-limiting examples of thyroid hormones and analogs thereof includeetoroxate, thyropropic acid and thyroxine.

Non-limiting examples of miscellaneous antihyperlipoproteinemics includeacifran, azacosterol, benfluorex, β-benzalbutyramide, carnitine,chondroitin sulfate, clomestrone, detaxtran, dextran sulfate sodium,5,8,11,14,17-eicosapentaenoic acid, eritadenine, furazabol, meglutol,melinamide, mytatrienediol, ornithine, γ-oryzanol, pantethine,pentaerythritol tetraacetate, α-phenylbutyramide, pirozadil, probucol(lorelco), β-sitosterol, sultosilic acid-piperazine salt, tiadenol,triparanol and xenbucin.

Non-limiting examples of an antiarteriosclerotic include pyridinolcarbamate.

In certain embodiments, administration of an agent that aids in theremoval or prevention of blood clots may be combined with administrationof a modulator, particularly in treatment of athersclerosis andvasculature (e.g., arterial) blockages. Non-limiting examples ofantithrombotic and/or fibrinolytic agents include anticoagulants,anticoagulant antagonists, antiplatelet agents, thrombolytic agents,thrombolytic agent antagonists or combinations thereof.

In certain aspects, antithrombotic agents that can be administeredorally, such as, for example, aspirin and wafarin (coumadin), arepreferred.

A non-limiting example of an anticoagulant include acenocoumarol,ancrod, anisindione, bromindione, clorindione, coumetarol, cyclocumarol,dextran sulfate sodium, dicumarol, diphenadione, ethyl biscoumacetate,ethylidene dicoumarol, fluindione, heparin, hirudin, lyapolate sodium,oxazidione, pentosan polysulfate, phenindione, phenprocoumon, phosvitin,picotamide, tioclomarol and warfarin.

Non-limiting examples of antiplatelet agents include aspirin, a dextran,dipyridamole (persantin), heparin, sulfinpyranone (anturane) andticlopidine (ticlid).

Non-limiting examples of thrombolytic agents include tissue plaminogenactivator (activase), plasmin, pro-urokinase, urokinase (abbokinase)streptokinase (streptase), anistreplase/APSAC (eminase).

In certain embodiments wherein a patient is suffering from a hemmorageor an increased likelyhood of hemmoraging, an agent that may enhanceblood coagulation may be used. Non-limiting examples of a bloodcoagulation promoting agent include thrombolytic agent antagonists andanticoagulant antagonists.

Non-limiting examples of anticoagulant antagonists include protamine andvitamine K1.

Non-limiting examples of thrombolytic agent antagonists includeamiocaproic acid (amicar) and tranexamic acid (amstat). Non-limitingexamples of antithrombotics include anagrelide, argatroban, cilstazol,daltroban, defibrotide, enoxaparin, fraxiparine, indobufen, lamoparan,ozagrel, picotamide, plafibride, tedelparin, ticlopidine and triflusal.

Non-limiting examples of antiarrhythmic agents include Class Iantiarrythmic agents (sodium channel blockers), Class II antiarrythmicagents (beta-adrenergic blockers), Class II antiarrythmic agents(repolarization prolonging drugs), Class IV antiarrhythmic agents(calcium channel blockers) and miscellaneous antiarrythmic agents.

Non-limiting examples of sodium channel blockers include Class IA, ClassIB and Class IC antiarrhythmic agents. Non-limiting examples of Class IAantiarrhythmic agents include disppyramide (norpace), procainamide(pronestyl) and quinidine (quinidex). Non-limiting examples of Class IBantiarrhythmic agents include lidocaine (xylocaine), tocainide(tonocard) and mexiletine (mexitil). Non-limiting examples of Class ICantiarrhythmic agents include encainide (enkaid) and flecainide(tambocor).

Non-limiting examples of a beta blocker, otherwise known as aβ-adrenergic blocker, a β-adrenergic antagonist or a Class IIantiarrhythmic agent, include acebutolol (sectral), alprenolol,amosulalol, arotinolol, atenolol, befunolol, betaxolol, bevantolol,bisoprolol, bopindolol, bucumolol, bufetolol, bufuralol, bunitrolol,bupranolol, butidrine hydrochloride, butofilolol, carazolol, carteolol,carvedilol, celiprolol, cetamolol, cloranolol, dilevalol, epanolol,esmolol (brevibloc), indenolol, labetalol, levobunolol, mepindolol,metipranolol, metoprolol, moprolol, nadolol, nadoxolol, nifenalol,nipradilol, oxprenolol, penbutolol, pindolol, practolol, pronethalol,propanolol (inderal), sotalol (betapace), sulfinalol, talinolol,tertatolol, timolol, toliprolol and xibinolol. In certain aspects, thebeta blocker comprises an aryloxypropanolamine derivative. Non-limitingexamples of aryloxypropanolamine derivatives include acebutolol,alprenolol, arotinolol, atenolol, betaxolol, bevantolol, bisoprolol,bopindolol, bunitrolol, butofilolol, carazolol, carteolol, carvedilol,celiprolol, cetamolol, epanolol, indenolol, mepindolol, metipranolol,metoprolol, moprolol, nadolol, nipradilol, oxprenolol, penbutolol,pindolol, propanolol, talinolol, tertatolol, timolol and toliprolol.

Non-limiting examples of an agent that prolong repolarization, alsoknown as a Class III antiarrhythmic agent, include amiodarone(cordarone) and sotalol (betap ace).

Non-limiting examples of a calcium channel blocker, otherwise known as aClass IV antiarrythmic agent, include an arylalkylamine (e.g.,bepridile, diltiazem, fendiline, gallopamil, prenylamine, terodiline,verapamil), a dihydropyridine derivative (felodipine, isradipine,nicardipine, nifedipine, nimodipine, nisoldipine, nitrendipine) apiperazinde derivative (e.g., cinnarizine, flunarizine, lidoflazine) ora micellaneous calcium channel blocker such as bencyclane, etafenone,magnesium, mibefradil or perhexiline. In certain embodiments a calciumchannel blocker comprises a long-acting dihydropyridine(nifedipine-type) calcium antagonist.

Non-limiting examples of miscellaneous antiarrhymic agents includeadenosine (adenocard), digoxin (lanoxin), acecainide, ajmaline,amoproxan, aprindine, bretylium tosylate, bunaftine, butobendine,capobenic acid, cifenline, disopyranide, hydroquinidine, indecainide,ipatropium bromide, lidocaine, lorajmine, lorcainide, meobentine,moricizine, pirmenol, prajmaline, propafenone, pyrinoline, quinidinepolygalacturonate, quinidine sulfate and viquidil.

Non-limiting examples of antihypertensive agents include sympatholytic,alpha/beta blockers, alpha blockers, anti-angiotensin II agents, betablockers, calcium channel blockers, vasodilators and miscellaneousantihypertensives.

Non-limiting examples of an alpha blocker, also known as an α-adrenergicblocker or an α-adrenergic antagonist, include amosulalol, arotinolol,dapiprazole, doxazosin, ergoloid mesylates, fenspiride, indoramin,labetalol, nicergoline, prazosin, terazosin, tolazoline, trimazosin andyohimbine. In certain embodiments, an alpha blocker may comprise aquinazoline derivative. Non-limiting examples of quinazoline derivativesinclude alfuzosin, bunazosin, doxazosin, prazosin, terazosin andtrimazosin.

In certain embodiments, an antihypertensive agent is both an alpha andbeta adrenergic antagonist. Non-limiting examples of an alpha/betablocker comprise labetalol (normodyne, trandate).

Non-limiting examples of anti-angiotension II agents include includeangiotensin converting enzyme inhibitors and angiotension II receptorantagonists. Non-limiting examples of angiotension converting enzymeinhibitors (ACE inhibitors) include alacepril, enalapril (vasotec),captopril, cilazapril, delapril, enalaprilat, fosinopril, lisinopril,moveltopril, perindopril, quinapril and ramipril. Non-limiting examplesof an angiotensin II receptor blocker, also known as an angiotension IIreceptor antagonist, an ANG receptor blocker or an ANG-II type-1receptor blocker (ARBS), include angiocandesartan, eprosartan,irbesartan, losartan and valsartan.

Non-limiting examples of a sympatholytic include a centrally actingsympatholytic or a peripherially acting sympatholytic. Non-limitingexamples of a centrally acting sympatholytic, also known as an centralnervous system (CNS) sympatholytic, include clonidine (catapres),guanabenz (wytensin) guanfacine (tenex) and methyldopa (aldomet).Non-limiting examples of a peripherally acting sympatholytic include aganglion blocking agent, an adrenergic neuron blocking agent, aβ-adrenergic blocking agent or a alpha1-adrenergic blocking agent.Non-limiting examples of a ganglion blocking agent include mecamylamine(inversine) and trimethaphan (arfonad). Non-limiting of an adrenergicneuron blocking agent include guanethidine (ismelin) and reserpine(serpasil). Non-limiting examples of a β-adrenergic blocker includeacenitolol (sectral), atenolol (tenormin), betaxolol (kerlone),carteolol (cartrol), labetalol (normodyne, trandate), metoprolol(lopressor), nadanol (corgard), penbutolol (levatol), pindolol (visken),propranolol (inderal) and timolol (blocadren). Non-limiting examples ofalpha1-adrenergic blocker include prazosin (minipress), doxazocin(cardura) and terazosin (hytrin).

In certain embodiments a cardiovasculator therapeutic agent may comprisea vasodilator (e.g., a cerebral vasodilator, a coronary vasodilator or aperipheral vasodilator). In certain preferred embodiments, a vasodilatorcomprises a coronary vasodilator. Non-limiting examples of a coronaryvasodilator include amotriphene, bendazol, benfurodil hemisuccinate,benziodarone, chloracizine, chromonar, clobenfurol, clonitrate, dilazep,dipyridamole, droprenilamine, efloxate, erythrityl tetranitrane,etafenone, fendiline, floredil, ganglefene, herestrolbis(β-diethylaminoethyl ether), hexobendine, itramin to sylate, khellin,lidoflanine, mannitol hexanitrane, medibazine, nicorglycerin,pentaerythritol tetranitrate, pentrinitrol, perhexiline, pimefylline,trapidil, tricromyl, trimetazidine, trolnitrate phosphate and visnadine.

In certain aspects, a vasodilator may comprise a chronic therapyvasodilator or a hypertensive emergency vasodilator. Non-limitingexamples of a chronic therapy vasodilator include hydralazine(apresoline) and minoxidil (loniten). Non-limiting examples of ahypertensive emergency vasodilator include nitroprusside (nipride),diazoxide (hyperstat IV), hydralazine (apresoline), minoxidil (loniten)and verapamil.

Non-limiting examples of miscellaneous antihypertensives includeajmaline, γ-aminobutyric acid, bufeniode, cicletainine, ciclosidomine, acryptenamine tannate, fenoldopam, flosequinan, ketanserin, mebutamate,mecamylamine, methyldopa, methyl 4-pyridyl ketone thiosemicarbazone,muzolimine, pargyline, pempidine, pinacidil, piperoxan, primaperone, aprotoveratrine, raubasine, rescimetol, rilmenidene, saralasin, sodiumnitrorus side, ticrynafen, trimethaphan camsylate, tyrosinase andurapidil.

In certain aspects, an antihypertensive may comprise an arylethanolaminederivative, a benzothiadiazine derivative, aN-carboxyalkyl(peptide/lactam) derivative, a dihydropyridine derivative,a guanidine derivative, a hydrazines/phthalazine, an imidazolederivative, a quanternary ammonium compound, a reserpine derivative or asuflonamide derivative.

Non-limiting examples of arylethanolamine derivatives includeamosulalol, bufuralol, dilevalol, labetalol, pronethalol, sotalol andsulfinalol.

Non-limiting examples of benzothiadiazine derivatives include althizide,bendroflumethiazide, benzthiazide, benzylhydrochlorothiazide,buthiazide, chlorothiazide, chlorthalidone, cyclopenthiazide,cyclothiazide, diazoxide, epithiazide, ethiazide, fenquizone,hydrochlorothizide, hydroflumethizide, methyclothiazide, meticrane,metolazone, paraflutizide, polythizide, tetrachlormethiazide andtrichlormethiazide.

Non-limiting examples of N-carboxyalkyl(peptide/lactam) derivativesinclude alacepril, captopril, cilazapril, delapril, enalapril,enalaprilat, fosinopril, lisinopril, moveltipril, perindopril, quinapriland ramipril.

Non-limiting examples of dihydropyridine derivatives include amlodipine,felodipine, isradipine, nicardipine, nifedipine, nilvadipine,nisoldipine and nitrendipine.

Non-limiting examples of guanidine derivatives include bethanidine,debrisoquin, guanabenz, guanacline, guanadrel, gu anazodine,guanethidine, guanfacine, guanochlor, guanoxabenz and guanoxan.

Non-limiting examples of hydrazines/phthalazines include budralazine,cadralazine, dihydralazine, endralazine, hydracarbazine, hydralazine,pheniprazine, pildralazine and todralazine.

Non-limiting examples of imidazole derivatives include clonidine,lofexidine, phentolamine, tiamenidine and tolonidine.

Non-limiting examples of quanternary ammonium compounds includeazamethonium bromide, chlorisondamine chloride, hexamethonium,pentacynium bis(methylsulfate), pentamethonium bromide, pentoliniumtartrate, phenactropinium chloride and trimethidinium methosulfate.

Non-limiting examples of reserpine derivatives include bietaserpine,deserpidine, rescinnamine, reserpine and syrosingopine.

Non-limiting examples of sulfonamide derivatives include ambuside,clopamide, furosemide, indapamide, quinethazone, tripamide and xipamide.

Vasopressors generally are used to increase blood pressure during shock,which may occur during a surgical procedure. Non-limiting examples of avasopressor, also known as an antihypotensive, include amezinium methylsulfate, angiotensin amide, dimetofrine, dopamine, etifelmin, etilefrin,gepefrine, metaraminol, midodrine, norepinephrine, pholedrine andsynephrine.

Non-limiting examples of agents for the treatment of congestive heartfailure include anti-angiotension II agents, afterload-preload reductiontreatment, diuretics and inotropic agents.

In certain embodiments, an animal patient that cannot tolerate anangiotension antagonist may be treated with a combination therapy. Suchtherapy may combine adminstration of hydralazine (apresoline) andisosorbide dinitrate (isordil, sorbitrate).

Non-limiting examples of a diuretic include a thiazide orbenzothiadiazine derivative (e.g., althiazide, bendroflumethazide,benzthiazide, benzylhydrochlorothiazide, buthiazide, chlorothiazide,chlorothiazide, chlorthalidone, cyclopenthiazide, epithiazide,ethiazide, ethiazide, fenquizone, hydrochlorothiazide,hydroflumethiazide, methyclothiazide, meticrane, metolazone,paraflutizide, polythizide, tetrachloromethiazide, trichlormethiazide),an organomercurial (e.g., chlormerodrin, meralluride, mercamphamide,mercaptomerin sodium, mercumallylic acid, mercumatilin dodium, mercurouschloride, mersalyl), a pteridine (e.g., furterene, triamterene), purines(e.g., acefylline, 7-morpholinomethyltheophylline, pamobrom,protheobromine, theobromine), steroids including aldosterone antagonists(e.g., canrenone, oleandrin, spironolactone), a sulfonamide derivative(e.g., acetazolamide, ambuside, azosemide, bumetanide, butazolamide,chloraminophenamide, clofenamide, clopamide, clorexolone,diphenylmethane-4,4′-disulfonamide, disulfamide, ethoxzolamide,furosemide, indapamide, mefruside, methazolamide, piretanide,quinethazone, torasemide, tripamide, xipamide), a uracil (e.g.,aminometradine, amisometradine), a potassium sparing antagonist (e.g.,amiloride, triamterene) or a miscellaneous diuretic such as aminozine,arbutin, chlorazanil, ethacrynic acid, etozolin, hydracarbazine,isosorbide, mannitol, metochalcone, muzolimine, perhexiline, ticrnafenand urea.

Non-limiting examples of a positive inotropic agent, also known as acardiotonic, include acefylline, an acetyldigitoxin, 2-amino-4-picoline,amrinone, benfurodil hemisuccinate, bucladesine, cerberosine,camphotamide, convallatoxin, cymarin, denopamine, deslanoside,digitalin, digitalis, digitoxin, digoxin, dobutamine, dopamine,dopexamine, enoximone, erythrophleine, fenalcomine, gitalin, gitoxin,glycocyamine, heptaminol, hydrastinine, ibopamine, a lanatoside,metamivam, milrinone, nerifolin, oleandrin, ouabain, oxyfedrine,prenalterol, proscillaridine, resibufogenin, scillaren, scillarenin,strphanthin, sulmazole, theobromine and xamoterol.

In particular aspects, an intropic agent is a cardiac glycoside, abeta-adrenergic agonist or a phosphodiesterase inhibitor. Non-limitingexamples of a cardiac glycoside includes digoxin (lanoxin) and digitoxin(crystodigin). Non-limiting examples of a β-adrenergic agonist includealbuterol, bambuterol, bitolterol, carbuterol, clenbuterol,clorprenaline, denopamine, dioxethedrine, dobutamine (dobutrex),dopamine (intropin), dopexamine, ephedrine, etafedrine,ethylnorepinephrine, fenoterol, formoterol, hexoprenaline, ibopamine,isoetharine, isoproterenol, mabuterol, metaproterenol, methoxyphenamine,oxyfedrine, pirbuterol, procaterol, protokylol, reproterol, rimiterol,ritodrine, soterenol, terbutaline, tretoquinol, tulobuterol andxamoterol. Non-limiting examples of a phosphodiesterase inhibitorinclude amrinone (inocor).

Antianginal agents may comprise organonitrates, calcium channelblockers, beta blockers and combinations thereof.

Non-limiting examples of organonitrates, also known asnitrovasodilators, include nitroglycerin (nitro-bid, nitrostat),isosorbide dinitrate (isordil, sorbitrate) and amyl nitrate (aspirol,vaporole).

2. Surgical Therapeutic Agents

In certain aspects, the secondary therapeutic agent may comprise asurgery of some type, which includes, for example, preventative,diagnostic or staging, curative and palliative surgery. Surgery, and inparticular a curative surgery, may be used in conjunction with othertherapies, such as the present invention and one or more other agents.

Such surgical therapeutic agents for vascular and cardiovasculardiseases and disorders are well known to those of skill in the art, andmay comprise, but are not limited to, performing surgery on an organism,providing a cardiovascular mechanical prostheses, angioplasty, coronaryartery reperfusion, catheter ablation, providing an implantablecardioverter defibrillator to the subject, mechanical circulatorysupport or a combination thereof. Non-limiting examples of a mechanicalcirculatory support that may be used in the present invention comprisean intra-aortic balloon counterpulsation, left ventricular assist deviceor combination thereof.

D. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Effects of Bucindolol and its Enantiomers on Nitric Oxide andPeroxynitrite Release from White, African American and Mexican AmericanEndothelial Cells: Comparison to Atenolol Methods

Donors and Cell Cultures. Human umbilical vein endothelial cells wereisolated into primary cultures from healthy female donors by Clonetics(San Diego, Calif.) and purchased as proliferating cells. All cellculture donors were healthy, with no pregnancy or prenatalcomplications. None of the donors took any drugs regularly and all werenonsmokers and consumed regular caloric/content diet. The cultured cellswere incubated in 95% air/5% CO₂ at 37° C. and passage by an enzymatic(trypsin) procedure. The confluent cells (4 to 5×10⁵ cells per 35-mmdish) were placed with minimum essential medium containing 3 mML-arginine and 0.1 mM (6R)-5,6,7,8-tetrahydrobiopterin (BH₄). Before theexperiments, the cells (from second or third passage) were rinsed twicewith Tyrode-HEPES buffer with 1.8 mM CaCl2. Bucindolol and itsenanatiomers were obtained from Arca Discovery (Denver, Colo.).

Measurement of NO and ONOO⁻ Levels. Measurement of NO was carried outwith electrochemical nanosensors (FIG. 1). Their design was based onpreviously developed and well-characterized chemically modifiedcarbon-fiber technology (Lvovich and Scheeline, 1997; Malinski and Taha,1992). Each of the nanosensors was constructed by depositing a sensingmaterial on the tip of a carbon fiber (length 4-5 μm, diameter 0.2-0.5μm). The fibers were sealed with nonconductive epoxy and electricallyconnected to copper wires with conductive silver epoxy. The inventorsused a conductive film of polymeric nickel (II) tetrakis(3-methoxy-4-hydroxyphenyl) porphyrin for the NO-sensor.

The NO nanosensors (diameter 1-2 μm) with a platinum wire (0.1 mm)counter electrode and saturated calomel reference electrode (SCE) wereapplied. Differential pulse voltammetry (DPV) and amperometry wereperformed with a computer-based Gamry VFP600 multichannel potentiostat.DPV was used to measure the basal NO concentrations, and amperometry wasused to measure changes in NO concentrations from its basal level withtime. The DPV current at the peak potential characteristic for NOoxidation (0.65 V) reduction was directly proportional to the localconcentrations of these compounds in the immediate vicinity of thesensor. Linear calibration curves (current vs. concentration) wereconstructed for each sensor from 10 nM to 3 μM before and aftermeasurements with aliquots of NO standard solutions, respectively. Thedetection limit of the sensors was 1.0 nM.

The quantification of each analyte (concentration in nmol/L) wasperformed using a maximum current from amperograms and standardcalibration curves. The reproducibility of measurements with nanosensorsis relatively high, as previously described (Lvovich and Scheeline,1997; Malinski and Taha, 1992). The NO nanosensor modules were loweredwith the help of a computer-controlled micromanipulator until it reachedthe surface of the cell membrane (a small piezoelectric signal, 0.1-0.2pA, of 1-3 milliseconds duration was observed at this point). Thesensors were slowly raised 5±2 μm from the surface of a singleendothelial cell.

The HUVEC preparation is stable over the course of these experimentswith the cells remaining viable and active responses to NO stimulationin culture for >24 hours. For robust statistical analysis, randomlyselected cells were used for each concentration and type of drug used inthese analyses.

Results

This study demonstrated reduced NO bioavailability in tissue frompatients in high-risk populations for hypertension, such as AfricanAmericans and Mexican Americans. As shown in FIG. 2,endothelial-dependent NO release from Mexican American and AfricanAmerican donors was 25% (305±28 nM, mean±S.D.) and 39% (251±27 nM)lower, respectively, than in non-Hispanic whites (409±23 nM), followingstimulation with a receptor-independent stimulus (1.0 μM CaI).

Treatment with the β₁-selective antagonist bucindolol, and especiallyits active enantiomer (S)-bucindolol, caused a dose-dependent increasein the capacity of the endothelium to generate NO (FIGS. 3-5).Pretreatment (6 hr) of the cells with bucindolol racemate (1.0 μM)increased NO release and enhanced endothelial activity in white donors(16% to 475±27 nM), Mexican American donors (5% to 321±28 nM), andAfrican American donors (22% to 306±25 nM).

In addition to reduced NO production, cells from these racial groupsshowed evidence of increased nitroxidative stress. The release of ONOO⁻from Mexican American and African American donors was higher by 57%(493±26 nM) and 37% (428±26 nM), respectively, than in non-Hispanicwhite donors (313±30 nM), following stimulation with CaI (FIG. 6).Treatment with the bucindolol (1.0 M), and especially its activeenantiomer (S)-bucindolol, caused a pronounced reduction innitroxidative stress. Bucindolol reduced ONOO⁻ levels by 19% (313±30 nMto 244±29 nM) in cells from non-Hispanic white donors, by 14% (493±26 nMto 422±27 nM) in cells from Mexican Americans and by 12% in cell fromAfrican Americans (428±26 nM to 378±30 nM) as shown in FIGS. 7-9. Thefavorable effects of bucindolol were much more apparent at 10.0 μM.

As compared to bucindolol racemate, a favorable effect on endothelialfunction was more pronounced with the active 1-selective enantiomer,(S)-bucindolol, while less activity was seen with (R)-bucindolol. Thedifferences in these enanatiomers were especially evident in cells fromnon-Hispanic white donors. Pretreatment (6 hr) of the cells with(S)-bucindolol (1.0 μM) increased NO release and enhanced endothelialactivity in white donors (35% to 553±35 nM), Mexican American donors(17% to 358±32 nM), and African American donors (27% to 318±16 nM). The(S)-bucindolol also reduced ONOO⁻ levels by 23% to 240±24 nM in cellsfrom non-Hispanic white donors as well as 15% to 419±34 nM in cells fromMexican Americans and 18% in cell from African Americans to 352±26 nM(FIGS. 7-9).

The most comprehensive measurement of endothelial function is the ratioof NO to ONOO⁻ following treatment with these agents. This studydemonstrated large differences in endothelial function in tissue fromhigher risk populations for hypertension, such as African Americans andMexican Americans (FIG. 10). The basis for these differences is notunderstood but may be due to genetic variability in the eNOS protein.Bucindolol racemate (1.0 μM) increased the NO/ONOO⁻ ratio in whitedonors (49%), Mexican American donors (23%), and African American donors(38%) as shown in FIGS. 11-13. An even greater effect was observed with(S)-bucindolol. Again, the favorable effects of bucindolol and itsenanatiomers were much more apparent at the higher dose (10.0 μM) by atleast two fold. The effect at the high concentration was especiallyevident in higher risks populations (African Americans, MexicanAmericans). In contrast, atenolol failed to produce an effect even atthe higher dose.

In summary, bucindolol had a dual effect on endothelial function byincreasing the capacity of cells to generate NO while simultaneouslyreducing ONOO⁻ production. The effect of bucindolol on endothelialfunction was dose-dependent and stereoselective; greater NObioavailability was associated with its active enantiomer,(S)-bucindolol. The basis for the benefit with bucindolol may be due tointeractions with novel receptor sites, such as the beta3 adrenergicreceptor, along its property of inhibition of oxidative stressmechanisms (e.g., NADPH oxidase). The favorable activity of bucindololon endothelial function was observed in three different racial groups,including African Americans, Mexican Americans and non-Hispanic whites.The activity of (S)-bucindolol was superior to bucindolol racemate andthe 1-selective blocker atenolol.

Example 2 Stereospecificity of Bucindolol Binding to Human β₁-AdrenergicReceptors

FIG. 14 gives the binding of S- and R-bucindolol in left ventricularmembranes prepared from one patient who had the β₁ 389 Arg/Arg receptor(FIG. 14A), and another with the Gly/Gly (FIG. 14B) genotype. As can beobserved, the S-isomer has much higher affinity, in both genotypes. Inaddition, the K_(i)s are similar for both the S- (0.5-0.6 nM) and R-(around 14-26 nM) isomers in the Arg/Arg and Gly/Gly preparations.Similar results were obtained in the presence of Gpp(NH)p.

Example 3 Separation of the R- and S-Enantiomers of BucindololHydrochloride

Chemistry. The usual procedure for the resolution of racemic aminederivatives involves fractional recrystallization of a mixture of thediastereomeric salts formed by combination of the base with an opticallyactive acid (e.g. d-tartaric). With bucindolol, however, this processproved too tedious and time consuming to be practical for medium scalework (5-25 g), and an alternate method was sought.

Racemic alcohols have been resolved by separation of the correspondingdiastereomeric carbamates produced by reaction with optically activearylalkyl isocyanates (Pirkle and Hoekstra, 1974; Pirkle and Hauske,1977a; Pirkle and Hauske, 1977b). Bucindolol, with a secondary hydroxylgroup in the side chain, would seem ideally suited for reaction withthis type of reagent. The reaction of bucindolol with(R)-(−)-(1-naphthyl)ethyl isocyanate, however, does not provide theanticipated carbamates but gives a pair of diastereomeric urea(Kolomiets et al., 1980) derivatives incorporating the basic nitrogen(Scheme 1). The greater reactivity of the basic nitrogen is not overcomeby steric hindrance from the bulky N-substituent. Resolution is achievedvia these ureas.

As the benzene solubility of one of the two isomeric ureas issignificantly less than the other, the initial separation becomesrelatively trivial and the less soluble isomer is obtained in excellentpurity. The residual material affords, by gravity column chromatography,the companion isomer in very satisfactory yield.

Generation of the individual optical antipodes is accomplished easily bystirring each diastereomer with hydrazine hydrate in ethanol solution.Treatment with pyruvic acid in this last step effectively removes theN-[(1-naphthyl)ethyl]hydrazinecarboxamide by-product and excesshydrazine.

Thus the resolution of bucindolol into its respective enantiomers isachieved via a facile new method that should be of general use for newβ-blocker molecules in the future.

Assignment of absolute configuration to the enantiomers of bucindolol(MJ 13105) is tentative, and based on the usual assignment ofS-configuration to the β-adrenergic aryloxypropanolamine enantiomer withnegative rotation (Danilewicz and Kemp, 1973).

Experimental. Melting points were determined using a Thomas-Hoovercapillary melting point apparatus and are uncorrected. Analytical valuesof carbon, hydrogen, and nitrogen are within 0.4% of theory and NMR, IR,and MS spectra are consistent with the assigned structures. Opticalrotation measurements were obtained on a Bendix-NPL 1169 automaticpolarimeter with digital readout.

Silica gel 60 (EM Reagents) was used for column chromatography.

The (g)-(−)-(1-naphthyl)ethyl isocyanate was purchased from AldrichChemical Co.

2-[2-Hydroxy-3-[[2-(1H-indol-3-yl)-1,1-dimethylethyl]amino]propoxy˜benzo-nitrile(MJ 13105 free base). A hot solution of bucindolol hydrochloride salt(100 g, 0.28 mol) and 2.5 l of H₂O was basified with a 10% solution ofNaOH. After cooling, the aqueous layer was decanted, and the residualgum rinsed with H₂O and crystallized from i-PrOH (500 mL) to provide 81g of MJ 13105 free base: mp 126-128° C.

The aqueous layer was allowed to stand overnight at 25° C., and theprecipitate was collected by filtration, washed with H₂O, and air driedovernight to give a 3.5 g second crop of MJ 13105 free base: mp 125-127°C.

(S), (R) and (R),(R)—N-[3-(2-Cyanophenoxy)-2-hydroxypropyl]-N-1,1-dimethyl-2-(1H-indol-3-yl)ethyl]-N′-[1-(1-naphthyl)ethyl]urea.A mixture of (R,S)-bucindolol (MJ 13105) free base (1.8 g, 0.0051 mol),(R)-(−)-1-(1-naphthyl)-ethyl isocyanate (1.0 g, 0.0051 mol), and benzene(100 mL) was stirred at 25° C. for 6 h. The white solid was removed byfiltration and air dried to give 1.24 g of (S),(R)—N-[3-(2-cyanophenoxy)-2-hydroxypropyl]-N-[1,1-dimethyl-2-(1H-indol-3-yl)ethyl]-N′-[1-(1-naphthyl)ethyl]urea:mp 167-168° C., one spot on TLC (silica gel;

$\left. {{{CH}_{2}{Cl}_{2}\text{/}{EtOAc}},{9\text{:}1}} \right),{{\lbrack\alpha\rbrack \frac{25}{D}} - {14{^\circ}\mspace{11mu} {\left( {{C\mspace{14mu} 0.5\%},{{CH}_{3}{OH}}} \right).}}}$

Anal. Calcd. for C₃₅H₃₆N₄O₃: C, 74.98; H, 6.48; N, 10.00. Found: C,74.89; H, 6.46; N, 9.74.

The filtrate was concentrated to dryness and the residue chromatographedon silica gel with CH₂Cl₂EtOAc (9:1) to give 0.70 g of(R),(R)—N-[3-(2-cyanophenoxy)-2-hydroxypropyl]-N-[1,1-dimethyl-2-(1H-indol-3-yl)ethyl]-N′-[1-(1-naphthyl)ethylurea as a foam:

${\lbrack\alpha\rbrack \frac{25}{D}} - {119{^\circ}\mspace{11mu} {\left( {{C{\mspace{11mu} \;}0.5\%},{{CH}_{3}{OH}}} \right).}}$

Anal. Calcd for C₃₅H₃₆N₄O₃-½ EtOAc: C,73.49; H, 6.67; N, 9.27. Found: C,73.29; H, 6.60; N, 9.18.

(S)-(−) and(R)-(+)-2-[2-Hydroxy-3-[[2-(1H-indol-3-yl)-1,1-dimethylethyl]-amino]propoxy]benzonitrilecyclamate (MJ 13105-163-997 and MJ 13105-163-998). The respectivediastereomer of the urea derivative was heated at reflux for 0.5 h inabsolute EtOH with five equivalents of 99% hydrazine hydrate. Afterevaporation of the solvent at reduced pressure, the residue wasdissolved in CH₃CN, and five equivalents of pyruvic acid were added. Thesolution was stirred at 25° C. overnight and concentrated at reducedpressure to give a residue that was dissolved in EtOAc. The EtOAcsolution was washed with three portions each of 1 N NaOH and H₂O, dried(anhyd. MgSO₄), filtered, and concentrated. One equivalent ofcyclohexanesulfamic acid was added to a solution of the weighed residueand absolute EtOH. After the mixture had cooled, the precipitated saltwas collected by filtration. Recrystallization from EtOH-(i-Pr)₂O-(DarcoG-60) gave the analytically pure samples of each isomer.

${(s)\text{-}( - )\text{-}{isomer}},{{mp}\mspace{14mu} 18O\text{-}181{^\circ}\mspace{14mu} {C.}},{{\lbrack\alpha\rbrack \frac{25}{D}} - {15.0{^\circ}\mspace{11mu} {\left( {{C\mspace{11mu} 1},{{CH}_{3}{OH}}} \right).}}}$

Anal. Calcd for C₂₂H₂₅N₃O₂ C₆H₁₃NO₃S: C, 61.98; H, 7.06; N, 10.33.Found: C, 62.12; H, 7.08; N, 10.31.

${(R)\text{-}( + )\text{-}{isomer}},{{{- {mp}}\mspace{14mu} 179\text{-}18{O{^\circ}}\mspace{14mu} {C.\; \lbrack\alpha\rbrack}\frac{25}{D}} + {15.5{^\circ}\mspace{11mu} {\left( {{C\mspace{11mu} 1},{{CH}_{3}{OH}}} \right).}}}$

Anal. Calcd for C₂₂H₂₅N₃O₂ C₆H₁₃NO₃S: C, 61.98; H, 7.06; N, 10.33.Found: C, 62-.07; H, 7.14; N, 10.11.

SCHEME 1

(R)-(+)-bucindolol

diastereomer (R), (R) and (S)-(−)-bucindolol

diastereomer (S), (R)

Example 4 Determination of Percent (R)- and (S)-Bucindolol Present inBucindolol Drug Substance and Drug Products by HPLC Summary

Chiral separation of (R)- and (S)-bucindolol is achieved by HPLC for thepurpose of determining the percent ratios present in drug substance ordrug products (tablets or capsules).

Equipment (as Stated Below, or Equivalent)

HPLC system (equipped with a UV detector capable of detection at 220 nm)

(Accompanying data systems are acceptable for quantitation)

Chiral Technologies, Inc. Chiralpak AD (4.6 mm×25 cm) column

0.2 μm filter, Acrodisc CR PTFE 0.2 μm Product No. 4225 25 mm

Reagents

Reagent Alcohol (Ethanol), HPLC grade

Diethylamine, reagent grade

Reference Standards

(R)-Bucindolol HCl Reference Standard and (S)-Bucindolol HCl ReferenceStandard or racemic Bucindolol HCl Reference Standard

Preparation of Solutions

Note: Prepare solutions in sufficient quantities to meet the needs ofthe analysis use appropriate proportions of solutions to maintain theratio of the final solution.

Mobile Phase

Add 1 mL of diethylamine (DEA) to every 500 mL of ethanol. Mix, filter,and degas under vacuum with sonication.

System Suitability Solution

Accurately weight 25±1 mg of (R)-bucindolol and 25±1 mg of(S)-bucindolol into the same 100 mL volumetric flask and dissolve inmobile phase. Transfer 3 mL to a 10 mL volumetric flask and dilute tovolume with mobile phase.

Alternatively, weigh approximately 50 mg of racemic bucindolol HClreference standard into a 100 mL volumetric flask and prepare as above.

Preparation of Samples

Prepare these solutions in duplicate.

Preparation of Drug Substance Samples

Accurately weigh 25±1 mg of the drug substance sample into a 50 mLvolumetric flask and dissolve in mobile phase. Transfer 3 mL to a 10 mLvolumetric flask and dilute to volume with mobile phase. Transfer the 10mL aliquot to a disposable syringe that is fitted with an 0.2 pm filter.Spend the first 2 mL to waste and collect the remaining portion foranalysis.

Preparation of Tablet Samples

Accurately weigh 20 tablets individually and determine the averagetablet weight. Grind the tablets as a composite. From the composite,accurately weigh the equivalent of one tablet into an appropriatevolumetric flask (V1 in Table 1). Dilute to volume with mobile phase andsonicate for 5 minutes. Transfer (T1) mL to an appropriate volumetricflask (V2) and dilute to volume with mobile phase (no further dilutionis required for some doses, as indicated by NA in the table). Transfer10 mL of the final solution to a disposable syringe that is fitted withan 0.2 μm filter. Spend the first 2 mL to waste and collect theremaining portion for analysis.

TABLE 1 Dose Initial Volume Transfer Volume Final Volume (mg) (V1) (mL)(T1) (mL) (V2) (mL) 3.0 25 NA NA 6.25 50 NA NA 12.5 100 NA NA 25.0 50 625 50.0 100 6 25 100 250 3 10

Preparation of Capsule Samples

Empty the contents of 20 capsules into a vial and mix to obtain acomposite. From the composite, accurately weigh 500 mg into anappropriate volumetric flask (V1 in Table 2). Dilute to volume withmobile phase and sonicate for 5 minutes. Transfer (T1) mL to anappropriate volumetric flask (V2) and dilute to volume with mobile phase(no further dilution is required for some doses, as indicated by NA inthe table). Transfer 10 mL of the final solution to a disposable syringethat is fitted with an 0.2 μm filter. Spend the first 2 mL to waste andcollect the remaining portion for analysis.

TABLE 2 Dose Initial Volume Transfer Volume Final Volume (mg) (V1) (mL)(T1) (mL) (V2) (mL) 3.0 25 NA NA 6.25 50 NA NA 12.5 100 NA NA 25.0 50 625 50.0 100 6 25 100 250 3 10

Chromatographic Conditions

Column Chiralpak AD (4.6 mm id × 25 cm) Column temperature ambientAutosampler Tray Temp. ambient Mobile Phase See section Mobile PhaseFlow rate 0.5 mL/min Needle Wash Mobile Phase section Injection Volume10 μL Injections per vial 2 Wavelength 220 nm Run time 15 min (may beadjusted as appropriate

System Suitability

System Interferences

Perform duplicate injections of mobile phase as a blank. No interferingpeaks or artifacts should be present in the blank chromatograms.

Relative Retention Times

Perform duplication injections of the system suitability solution. Usingthe second injection, report the relative retention times of eachbucindolol peak. The relative retention times of (R)- and (S)-bucindololshould be approximately 1.00 and 1.23, respectively.

Peak Tailing

Using the same injection as for Relative Retention Times above,calculate the tailing factors for both bucindolol peaks as follows:

$T = \frac{W}{2 \times F}$

-   -   where: T=tailing factor        -   W=peak width at 5% of peak height        -   F=width of line from peak start to the retention time at 5%            of peak height    -   The tailing factors for both peaks should be ≤1.5

System Suitability

Perform duplicate injections of each sample.

Calculations

For all substance and product samples, calculate the percent (R)- andpercent (S)-bucindolol as follows:

% (R)-bucindolol=(peak area (R)×100%/(peak area (R)+peak area (S))

% (S)-bucindolol=(peak area (S)×100%/(peak area (R)+peak area (S))

Example 5 Preparations of (S)-Bucindolol

Reagents MW Density Amount Units mmol Eq. Source 221.62 — 14 g 631.71 1Ald/02807CE (S)Glycidol 74.08 1.116 4.19 ml 631.71 1 Ald/2007DE Et₃N101.19 0.720 8.878 ml 6.3171 1 Toluene 150 ml

Procedure:

To a round-bottom flask 4.19 ml (S)-glycidol, 8.878 ml of Et₃N and 150ml of Toluene was added. The reaction mixture was stirred in a N₂ atm.The reaction mixture was cooled to −10° C. Then Nitrosulfaryl chloridewas added in 3 lots. The reaction mixture was stirred for two hours.After the completion of the reaction, water was added to the reactionmixture. The compound was taken up in EtOAc. The EtOAc layer was washedwith brine. The EtOAc layer was dried and concentrated. The compound waspurified by column chromatography.

Theoretical Yield: 16.376 g

% Yield: 93%

Yield obtained: 15.140 g

¹H NMR: verified

Reagents MW Density Amount Units mmol Eq. 2-Cyanophenol 119.12 — 1 g8.394 1 Nolylate 259.24 — 2.17 g 8.394 1 K₂CO₃ 138.21 3.48 g 25.182 3acetone 50 ml (HPLC grade)

Procedure:

2-Cyanophenol was taken up in acetone. To that K₂CO₃ was added. Thereaction mixture was refluxed for 30 min. Then the reaction mixture wascooled to room temperature. Nolylate was added and again refluxed. Thereaction was monitored by HPLC. After the completion of the reaction,K₂CO₃ was then filtered/removed using scintered glass crucible. Thefiltrate was concentrated and purified by column chromatography (30%EtOAc in hexanes).

Theoretical Yield: 1.470 g

Yield Obtained: 1.4 g

¹H NMR: verified

Reagents MW Density Amount Units mmol Eq. Epoxide 175.18 — 7 g 39.958 1Amine 188.269 — 7.52 g 39.958 1 Ethanol 125 ml

Procedure:

Epoxide and amine dissolved in EtOH and refluxed. After the completionof the reaction, the ethanol was removed. The residue was purified bycolumn chromatography.

Theoretical Yield: 14.52 g

Yield Obtained: 5.5 g, HPLC purity 100% (1^(st) reaction)

-   -   6.5 g HPLC purity 98% (2^(nd) reaction)

¹H NMR: verified for both

Reagents MW Density Amount Units mmol Eq. SM 363.45 — 5.5 g 15.132 1 HClin 1.5M 10.08 ml 15.132 1 ether Diethyl 50 ml ether

Procedure:

The starting material was dissolved in diethyl ether and to that 1.5 MHCl was added and allowed to stir for 2 hours. After that diethyl etherwas removed. The resulting solid was dried.

Theoretical Yield: 6.06 g

Yield Obtained: 5.750 g

% Yield: 95%

Chiral HPLC purity: 100%

HPLC purity: 99%

¹H NMR: verified

Reagents MW Density Amount Units mmol Eq. Source 221.62 — 15 g 67.683 1AW/12007DE R-glycidol 74.08 1.116 4.49 ml 67.683 1 Ald/02020BH Et₃N101.19 0.720 9.512 ml 67.683 1 Toluene

Procedure: As in IN-SPL-C-11

To R-glycidol, toluene and Et₃N were added. Then Nitrosulfonyl chloridewas added in lots at −10° C. and stirred for 2 hours. FollowingEtOAc/water work up, the residue was purified by column chromatography.

Theoretical Yield: 17.54 g

Yield Obtained: 15.5 g

% Yield: 88%

¹H NMR: verified

Reagents MW Density Amount Units mmol Eq. Source 2-Cyanophenol 119.12 —6.89 g 57.86 1 AM R1/290328 Nolylate 259.24 — 15.0 g 57.86 1 K₂CO₃138.21 — 23.9 g 173.58 3 Acetone 200 ml

Procedure:

2-Cyanophenol in acetone and K₂CO₃ were heated at reflux for 30 min.Then cooled Nolylate was added and heated to reflux. As in Reaction 2,the reaction was monitored by HPLC. After the completion of the reactionK₂CO₃ filtered/removed. The filtrate was concentrated and purified bycolumn chromatography.

Theoretical Yield: 10.13 g

Yield Obtained: 7.3 g

% Yield: 72%

¹H NMR: verified

Reagents MW Density Amount Units mmol Eq. Epoxide 175.18 — 7 g 39.958 1Amine 188.269 — 7.52 g 39.958 1 Ethanol 150 ml

Procedure:

The Epoxide and amine, each in Ethanol, were heated to reflux. ThenEthanol was removed after the completion of reaction and purified bycolumn chromatography.

Theoretical Yield: 14.523 g

Yield Obtained: 10.5 g

% Yield: 72%

Reagents MW Density Amount Units mmol Eq. Starting 363.453 — 10 g 27.511 material HCl in 1.5M 18.34 ml 27.51 1 Ether Diethyl 200 ml ether

Procedure:

Starting material was dissolved in diethyl ether. To that 1.5 M HCl wasadded and allowed to stir for 2 hours. Then ether was removed. Theresulting solid was dried.

Theoretical Yield: 11.030 g

Yield Obtained: 10.5 g

% Yield: 95%

Chiral HPLC purity: 100%

HPLC Purity: 99%

¹H NMR: verified

Example 6 Characterization Data of (R)- and (S)-Bucindolol

See FIGS. 15 and 16 for ¹H NMR spectra of (R)- and (S)-bucindolol.

(R)-Bucindolol:

TEST RESULT REFERENCE Appearance Light green solid 300 MHz ¹H NMRConsistent-Attached Spectrum (DMSO-d₆) Mass Spectrum ESI, m/z 364 [M +H]⁺, Attached Chiral HPLC Analysis >99% (area %), >99% ee, CHIRALPAK ADColumn, Detector @ 280 nm, Attached Optical Rotation [α]^(22.9) _(D) +15.5° (c 1.00, Methanol)

(S)-Bucindolol

TEST RESULT/REFERENCE Appearance White solid 300 MHz ¹H NMRConsistent-Attached Spectrum (DMSO-d₆) Mass Spectrum ESI, m/z 364 [M +H]⁺, Attached Chiral HPLC Analysis >99% (area %), >99% ee, CIHRALPAK ADColumn, Detector @ 280 nm, Attached Optical Rotation [α]^(22.7) _(D) −15.9° (c 1.00, Methanol)

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of some embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1.-46. (canceled)
 47. A method of increasing the capacity of a cell togenerate NO, comprising contacting said cell with a compositioncomprising a pharmaceutically acceptable carrier and bucindololsubstantially free of its R-stereoisomer.
 48. The method of claim 47,wherein the cell is an endothelial cell, an epithelial cell, or a stemcell.
 49. The method of claim 47, wherein the cell is an endothelialcell.
 50. The method of claim 47, wherein the cell is in a patient. 51.The method of claim 47, wherein the cell is in an animal.
 52. The methodof claim 51, wherein the animal is a mouse, a rat, a rabbit, a cat, adog, a horse, a sheep, a goat, a cow, or a primate. 53.-62. (canceled)